Compositions, methods and kits to detect adenovirus, metapneumovirus and/or rhinovirus nucleic acids

ABSTRACT

The disclosed disclosure is related to methods, compositions, and kits for targeting Adenovirus, Metapneumovirus, and/or Rhinovirus nucleic acid. Compositions include amplification oligomers and/or detection probe oligomers. Kits and methods comprise at least one of these oligomers. Methods include uniplex and multiplex amplification and detection reactions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority of U.S. provisionalapplication No. 62/476,753, which was filed on Mar. 25, 2017, and whichis incorporated herein by reference.

FIELD

The present disclosure relates to the detection of infectious agents,more specifically to the detection of Adenovirus, Metapneumovirus,and/or Rhinovirus. Compositions, methods and kits are described for theamplification and/or detection of Adenovirus, Metapneumovirus, and/orRhinovirus by using in vitro nucleic acid amplification techniques.

INTRODUCTION

Infectious diseases, whether or bacterial, viral, or other origin,present acute and chronic challenges to human health. Many commoninfections affect the respiratory tract. Respiratory tract diseases areprevalent in patients of all ages, although often are more serious inthe very young and the very old. Viruses include DNA viruses and RNAviruses.

Adenovirus (Adeno or Adv) may cause infections in a number of differentorgans including the gastrointestinal tract, the upper respiratory tractand the eyes. In individuals with a properly functioning immune system,Adenovirus infections are not typically associated with life-threateningdisease. However, Adenovirus can cause serious infection inimmuno-compromised patients—such as HIV-positive individuals and inpatients receiving bone marrow transplants. More than 50 different humanAdenovirus serotypes have been identified. On the basis of variousproperties of Adenovirus, they have been divided into six majorsubgroups (subgenera or species A-F), with recent literature pointingtowards the presence of a seventh serotype.

Early approaches for detecting Adeno detection relied mainly onserological tests and cell culture. In immunosuppressed patients,however, the use of serological tests is limited due to the impairedimmune response, and evaluation of positive cultures is a relativelyslow method. The introduction of PCR-based assays has provided newmethods for the rapid, specific and sensitive detection of Adenovirus.Many of these diagnostic approaches, however, do not effectively coverall Adenovirus serotypes or use low stringency conditions to permitdetection of the genetically highly diverse adenoviruses.

The homology of adenovirus DNA sequences between different species islow. Even conserved regions within the Adenovirus genome display onlylimited homology between adenoviruses from different species. In manyinstances, considerable differences in DNA sequence even exist betweenserotypes belonging to the same species. These facts underscore thedifficulty to develop molecular tests that facilitate reliable screeningfor Adenovirus infections with the required broad specificity.

The human metapneumovirus (hMPV) was isolated for the first time in 2001and is now recognized to be the second major cause of acute respiratorytract disease in infants and adults. It is estimated that it infectsover 50% of infants by two years of age and almost all children by fiveyears. hMPV accounts for roughly 5 to 15% of respiratory disease inhospitalized young children (Alto, 2004, The Journal of the AmericanBoard of Family Practice/American Board of Family Practice 17:466-469;Williams et al., 2004, N Engl J Med 350:443-450). Infection with hMPV isa significant burden of disease in at-risk premature infants, chroniclung disease of prematurity, congestive heart disease, andimmunodeficiency (Martino et al., 2005, Biology of Blood and MarrowTransplantation: Journal of the American Society for Blood and MarrowTransplantation 11:781-796).

There are two distinct genetic lineages that have been established forhMPV and are designated as subtypes A and B. These lineages have furtherbeen divided into subgroups A1, A2, B1 and B2, as determined byperforming phylogenetic analysis of sequence data, most often utilizingthe fusion protein and G glycoprotein gene. No significant differenceshave been observed among patients infected with different subgroups ofhMPV in terms of clinical manifestations (Wei, H., Tsao, K., Huang, C.,Huang, Y., Lin, T. J Microbiol Immunol Infect. 2012 Sep. 26. pii:S1684-1182(12)00151-X). Although information on the mode of transmissionand virulence is not definitive, hMPV is likely spread by similar meansas common respiratory viruses such as influenza. hMPV has been shown toco-infect with other respiratory pathogens. hMPV appears to provide onlypartial immunity following infection, likely due to the variety ofstrains and subtypes that circulate during any given season, and canre-infect individuals potentially leading to repeated episodes ofillness. Infections occur mainly during late winter and early spring andthe prevalence of each subtype of hMPV varies, seemingly both from yearto year, and by location. Likewise, the overall incidence of hMPV canvary from year to year and its prevalence has been reported to rangefrom 2 to 26% in patients with symptoms of respiratory infection.

Human rhinoviruses (HRVs) are the most frequent cause of acute upperrespiratory tract infections in humans and are usually associated withthe common cold. Common colds caused by HRV occur throughout the year,with peaks of incidence in the autumn and spring, are one of the mainreasons for absences from work and school, which have major economicimpact Rhinoviruses can also cause lower respiratory tract infectionsresulting in severe disease in children, in the elderly and inimmunosuppressed patients.

The HRVs, which include over 100 different serotypes, are small,non-enveloped, positive (+)-strand RNA viruses. HRVs are one of the sixgenera of Picornaviridae, which also includes enteroviruses (EVs).Reverse transcription-polymerase chain reaction (RT-PCR) has beendeveloped in the past few years for the detection of the HRVs inclinical specimens (see, e.g., Billaud et al. (2003) J. Virol. Methods108: 223-228; Blomqvist et al. (1999) J. Clin, Microbiol. 37:2813-2816;Kares et al. (2003) J Clin Virol. 2004 February; 29(2):99-104; Loens etal. (2003) J. Clin. Microbiol. 41: 1971-1976; Savolainen et al. (2002)J. General Virol. 83: 333-340; Steininger et al. (2001) J. Clin.Microbiol. 39: 129-133). Most of these RT-PCR methods take advantage ofthe conserved sequences in the 5′ noncoding region of the picornavirusgenome.

The ability to detect HRV specifically—and particularly avoiding falsepositives that can result due to the relatedness of HRV and EV—isimportant to both diagnosis and selection of appropriate availabletherapy. Specific assays for HRV are also important for development ofnew drugs. For example, it is critical for clinical trial design thatthe participants be correctly identified as having an HRV infectionwhere the trial is designed to evaluate a drug for use in treatment ofHRV infections. Moreover, in other clinical trials, it may be importantto exclude individuals infected with HRV. Further, the HRV detectionassays must be simple to perform, provide easily interpreted results,and be relatively inexpensive to make them practical for use.

Conventional methods of differentiation of HRVs from EVs has been doneeither by virus neutralization assay, by selection with HRV-specificprimer pairs, by distinguishing the amplification products of the twoviruses based on differences in size, by sequencing the amplificationproducts and comparing the sequence to known HRV and EV sequences, or byhybridization using HRV or EV-specific probes. These approaches can betime-consuming, expensive, and/or require a skilled technician who hasexperience in interpreting assay results accurately.

There remains a need in the field for a molecular based assay to permitthe rapid, sensitive and specific detection of multiple adenovirusserotypes. There also remains a need in the field for the rapid,sensitive and specific detection of the multiple subtypes and subgroupsof hMPV. There further remains a need in the field for methods fordetecting RVs in a manner that is rapid, sensitive and specific,particularly with respect to the ability to distinguish an RV from anEV.

SUMMARY

It is an object of the present disclosure to provide methods,compositions and kits that can be used to specifically amplify and/ordetect with high sensitivity one or more of an Adenovirus, a hMPV, and aHRV nucleic acid. Advantageously, the methods, compositions and kits maybe used to specifically detect with high sensitivity many (eg. 5 ormore, 10 or more, 20 or more, 30 or more, 40 or more or 50 or more), orall known serotypes and subgroups of adenovirus, hMPV and/or HRV.

1. A composition or kit comprising at least first and secondamplification oligomers, wherein:

-   -   the first amplification oligomer and second amplification        oligomer are configured to amplify an Adenovirus amplicon of at        least about 50 nucleotides in length comprising at least one        Adenovirus position located in the range of nucleotide positions        selected from 52 to 74 and/or 76 to 99 and/or 40 to 56 and/or 65        to 87 and/or 1 to 18 and/or 7 to 23 and/or 28 to 45 and/or 27 to        45 and/or 26 to 45 and/or 139 to 155 and/or 103 to 123 and/or        159 to 175 and/or 83 to 99 and/or 83 to 98 of SEQ ID No. 47.

2. A composition or kit comprising at least first and secondamplification oligomers, wherein:

-   -   the first amplification oligomer and second amplification        oligomer are configured to amplify a Metapneumovirus amplicon of        at least about 50 nucleotides in length comprising at least one        Metapneumovirus position located in the range of nucleotide        positions selected from 966 to 1147 of SEQ ID NO:150, and/or        nucleotides 844 to 1027 of SEQ ID NO:159, and/or 1000 to 1040 of        SEQ ID NO:150, and/or 880 to 915 of SEQ ID NO:159, and/or 1027        to 1080 of SEQ ID NO:150, and/or 913 to 958 of SEQ ID NO:159,        and/or 1073 to 1115 of SEQ ID NO:150, and/or 953 to 995 of SEQ        ID NO:159.

3. A composition or kit comprising at least first and secondamplification oligomers, wherein:

-   -   the first amplification oligomer and second amplification        oligomer are configured to amplify a Rhinovirus amplicon of at        least about 50 nucleotides in length comprising at least one        Rhinovirus position located in the range of nucleotide positions        selected from 230 to 556 of SEQ ID NO:120, and/or 199 to 525 of        SEQ ID NO:101, and/or 80 to 410 of SEQ ID NO:76, and/or 263 to        303 of SEQ ID NO:120, and/or 231 to 264 of SEQ ID NO:101, and/or        106 to 156 of SEQ ID NO:76, and/or 312 to 346 of SEQ ID NO:120,        and/or 279 to 314 of SEQ ID NO:101, and/or 455 to 506 of SEQ ID        NO:76, and/or 480 to 533 of SEQ ID NO:120, and/or 455 to 506 of        SEQ ID NO:101, and/or 338 to 397 of SEQ ID NO:76.

4. A composition or kit comprising at least first and secondamplification oligomers configured for two or more target acids,wherein:

-   -   (A) for a first target nucleic acid the first amplification        oligomer and second amplification oligomer are configured to        amplify an Adenovirus amplicon of at least about 50 nucleotides        in length comprising at least one Adenovirus position located in        the range of nucleotide positions selected from 52 to 74 and/or        76 to 99 and/or 40 to 56 and/or 65 to 87 and/or 1 to 18 and/or 7        to 23 and/or 28 to 45 and/or 27 to 45 and/or 26 to 45 and/or 139        to 155 and/or 103 to 123 and/or 159 to 175 and/or 83 to 99        and/or 83 to 98 of SEQ ID No. 47; and    -   (B) for a second target nucleic acid;        -   (i) the first amplification oligomer and second            amplification oligomer are configured to amplify a            Metapneumovirus amplicon of at least about 50 nucleotides in            length comprising at least one Metapneumovirus position            located in the range of nucleotide positions selected from            966 to 1147 of SEQ ID NO:150, and/or nucleotides 844 to 1027            of SEQ ID NO:159, and/or 1000 to 1040 of SEQ ID NO:150,            and/or 880 to 915 of SEQ ID NO:159, and/or 1027 to 1080 of            SEQ ID NO:150, and/or 913 to 958 of SEQ ID NO:159, and/or            1073 to 1115 of SEQ ID NO:150, and/or 953 to 995 of SEQ ID            NO:159; or        -   (ii) the first amplification oligomer and second            amplification oligomer are configured to amplify a            Rhinovirus amplicon of at least about 50 nucleotides in            length comprising at least one Rhinovirus position located            in the range of nucleotide positions selected from 230 to            556 of SEQ ID NO:120, and/or 199 to 525 of SEQ ID NO:101,            and/or 80 to 410 of SEQ ID NO:76, and/or 263 to 303 of SEQ            ID NO:120, and/or 231 to 264 of SEQ ID NO:101, and/or 106 to            156 of SEQ ID NO:76, and/or 312 to 346 of SEQ ID NO:120,            and/or 279 to 314 of SEQ ID NO:101, and/or 455 to 506 of SEQ            ID NO:76, and/or 480 to 533 of SEQ ID NO:120, and/or 455 to            506 of SEQ ID NO:101, and/or 338 to 397 of SEQ ID NO:76.

5. A composition or kit comprising at least first and secondamplification oligomers configured for two or more target acids,wherein:

-   -   (A) for a first target nucleic acid the first amplification        oligomer and second amplification oligomer are configured to        amplify a Metapneumovirus amplicon of at least about 50        nucleotides in length comprising at least one Metapneumovirus        position located in the range of nucleotide positions selected        from 966 to 1147 of SEQ ID NO:150, and/or nucleotides 844 to        1027 of SEQ ID NO:159, and/or 1000 to 1040 of SEQ ID NO:150,        and/or 880 to 915 of SEQ ID NO:159, and/or 1027 to 1080 of SEQ        ID NO:150, and/or 913 to 958 of SEQ ID NO:159, and/or 1073 to        1115 of SEQ ID NO:150, and/or 953 to 995 of SEQ ID NO:159; and    -   (B) for a second target nucleic acid;        -   (i) the first amplification oligomer and second            amplification oligomer are configured to amplify an            Adenovirus amplicon of at least about 50 nucleotides in            length comprising at least one Adenovirus position located            in the range of nucleotide positions selected from 52 to 74            and/or 76 to 99 and/or 40 to 56 and/or 65 to 87 and/or 1 to            18 and/or 7 to 23 and/or 28 to 45 and/or 27 to 45 and/or 26            to 45 and/or 139 to 155 and/or 103 to 123 and/or 159 to 175            and/or 83 to 99 and/or 83 to 98 of SEQ ID No. 47; or        -   (ii) the first amplification oligomer and second            amplification oligomer are configured to amplify a            Rhinovirus amplicon of at least about 50 nucleotides in            length comprising at least one Rhinovirus position located            in the range of nucleotide positions selected from 230 to            556 of SEQ ID NO:120, and/or 199 to 525 of SEQ ID NO:101,            and/or 80 to 410 of SEQ ID NO:76, and/or 263 to 303 of SEQ            ID NO:120, and/or 231 to 264 of SEQ ID NO:101, and/or 106 to            156 of SEQ ID NO:76, and/or 312 to 346 of SEQ ID NO:120,            and/or 279 to 314 of SEQ ID NO:101, and/or 455 to 506 of SEQ            ID NO:76, and/or 480 to 533 of SEQ ID NO:120, and/or 455 to            506 of SEQ ID NO:101, and/or 338 to 397 of SEQ ID NO:76.

6. A composition or kit comprising at least first and secondamplification oligomers configured for two or more target acids,wherein:

-   -   (A) for a first target nucleic acid the first amplification        oligomer and second amplification oligomer are configured to        amplify a Rhinovirus amplicon of at least about 50 nucleotides        in length comprising at least one Rhinovirus position located in        the range of nucleotide positions selected from 230 to 556 of        SEQ ID NO:120, and/or 199 to 525 of SEQ ID NO:101, and/or 80 to        410 of SEQ ID NO:76, and/or 263 to 303 of SEQ ID NO:120, and/or        231 to 264 of SEQ ID NO:101, and/or 106 to 156 of SEQ ID NO:76,        and/or 312 to 346 of SEQ ID NO:120, and/or 279 to 314 of SEQ ID        NO:101, and/or 455 to 506 of SEQ ID NO:76, and/or 480 to 533 of        SEQ ID NO:120, and/or 455 to 506 of SEQ ID NO:101, and/or 338 to        397 of SEQ ID NO:76; and    -   (B) for a second target nucleic acid;        -   (i) the first amplification oligomer and second            amplification oligomer are configured to amplify an            Adenovirus amplicon of at least about 50 nucleotides in            length comprising at least one Adenovirus position located            in the range of nucleotide positions selected from 52 to 74            and/or 76 to 99 and/or 40 to 56 and/or 65 to 87 and/or 1 to            18 and/or 7 to 23 and/or 28 to 45 and/or 27 to 45 and/or 26            to 45 and/or 139 to 155 and/or 103 to 123 and/or 159 to 175            and/or 83 to 99 and/or 83 to 98 of SEQ ID No. 47; or        -   (ii) the first amplification oligomer and second            amplification oligomer are configured to amplify a            Metapneumovirus amplicon of at least about 50 nucleotides in            length comprising at least one Metapneumovirus position            located in the range of nucleotide positions selected from            966 to 1147 of SEQ ID NO:150, and/or nucleotides 844 to 1027            of SEQ ID NO:159, and/or 1000 to 1040 of SEQ ID NO:150,            and/or 880 to 915 of SEQ ID NO:159, and/or 1027 to 1080 of            SEQ ID NO:150, and/or 913 to 958 of SEQ ID NO:159, and/or            1073 to 1115 of SEQ ID NO:150, and/or 953 to 995 of SEQ ID            NO:159.

7. A composition or kit comprising at least first and secondamplification oligomers configured for three or more target acids,wherein:

-   -   (A) for a first target nucleic acid the first amplification        oligomer and second amplification oligomer are configured to        amplify an Adenovirus amplicon of at least about 50 nucleotides        in length comprising at least one Adenovirus position located in        the range of nucleotide positions selected from 52 to 74 and/or        76 to 99 and/or 40 to 56 and/or 65 to 87 and/or 1 to 18 and/or 7        to 23 and/or 28 to 45 and/or 27 to 45 and/or 26 to 45 and/or 139        to 155 and/or 103 to 123 and/or 159 to 175 and/or 83 to 99        and/or 83 to 98 of SEQ ID No. 47; and    -   (B) for a second target nucleic acid the first amplification        oligomer and second amplification oligomer are configured to        amplify a Metapneumovirus amplicon of at least about 50        nucleotides in length comprising at least one Metapneumovirus        position located in the range of nucleotide positions selected        from 966 to 1147 of SEQ ID NO:150, and/or nucleotides 844 to        1027 of SEQ ID NO:159, and/or 1000 to 1040 of SEQ ID NO:150,        and/or 880 to 915 of SEQ ID NO:159, and/or 1027 to 1080 of SEQ        ID NO:150, and/or 913 to 958 of SEQ ID NO:159, and/or 1073 to        1115 of SEQ ID NO:150, and/or 953 to 995 of SEQ ID NO:159; and    -   (C) for a third target nucleic acid the first amplification        oligomer and second amplification oligomer are configured to        amplify a Rhinovirus amplicon of at least about 50 nucleotides        in length comprising at least one Rhinovirus position located in        the range of nucleotide positions selected from 230 to 556 of        SEQ ID NO:120, and/or 199 to 525 of SEQ ID NO:101, and/or 80 to        410 of SEQ ID NO:76, and/or 263 to 303 of SEQ ID NO:120, and/or        231 to 264 of SEQ ID NO:101, and/or 106 to 156 of SEQ ID NO:76,        and/or 312 to 346 of SEQ ID NO:120, and/or 279 to 314 of SEQ ID        NO:101, and/or 455 to 506 of SEQ ID NO:76, and/or 480 to 533 of        SEQ ID NO:120, and/or 455 to 506 of SEQ ID NO:101, and/or 338 to        397 of SEQ ID NO:76.

8. The composition or kit of any one of claims 1 to 3, wherein the firstamplification oligomer comprises a nucleic acid sequences that containsat least one 5-Me-dC, at least one non-Watson Crick base, at least onedegenerate base, or a combination thereof.

9. The composition or kit of any of claim 1 to 3 or 8, wherein thesecond amplification oligomer comprises a nucleic acid sequence thatcontains at least one 5-Me-dC, or at least one non-Watson Crick base, orat least one degenerate base, or a combination thereof.

10. The composition or kit of claim 4 or claim 7, wherein the firstamplification oligomer configured to amplify an Adenovirus ampliconcomprises a nucleic acid sequences that contains at least one 5-Me-dC,or at least one non-Watson Crick base, or at least one degenerate base,or a combination thereof, and/or wherein the second amplificationoligomer configured to amplify an Adenovirus amplicon comprises anucleic acid sequences that contains at least one 5-Me-dC, or at leastone non-Watson Crick base, or at least one degenerate base, or acombination thereof.

11. The composition or kit of claim 5 or claim 6, wherein the secondtarget nucleic acid is an Adenovirus target nucleic acid and wherein thefirst amplification oligomer configured to amplify an Adenovirusamplicon comprises a nucleic acid sequences that contains at least one5-Me-dC, at least one non-Watson Crick base, at least one degeneratebase, or a combination thereof, or wherein the second amplificationoligomer configured to amplify an Adenovirus amplicon comprises anucleic acid sequences that contains at least one 5-Me-dC, at least onenon-Watson Crick base, at least one degenerate base, or a combinationthereof, or both.

12. The composition or kit of claim 5 or claim 7, wherein the firstamplification oligomer configured to amplify a Metapneumovirus ampliconcomprises a nucleic acid sequences that contains at least one 5-Me-dC,at least one non-Watson Crick base, at least one degenerate base, or acombination thereof, or wherein the second amplification oligomerconfigured to amplify an Metapneumovirus amplicon comprises a nucleicacid sequences that contains at least one 5-Me-dC, at least onenon-Watson Crick base, at least one degenerate base, or a combinationthereof, or both.

13. The composition or kit of claim 4 or claim 6, wherein the secondtarget nucleic acid is an Metapneumovirus target nucleic acid andwherein the first amplification oligomer configured to amplify anMetapneumovirus amplicon comprises a nucleic acid sequences thatcontains at least one 5-Me-dC, at least one non-Watson Crick base, atleast one degenerate base, or a combination thereof, or wherein thesecond amplification oligomer configured to amplify an Metapneumovirusamplicon comprises a nucleic acid sequences that contains at least one5-Me-dC, at least one non-Watson Crick base, at least one degeneratebase, or a combination thereof, or both.

14. The composition or kit of claim 6 or claim 7, wherein the firstamplification oligomer configured to amplify a Rhinovirus ampliconcomprises a nucleic acid sequences that contains at least one 5-Me-dC,at least one non-Watson Crick base, at least one degenerate base, or acombination thereof, or wherein the second amplification oligomerconfigured to amplify an Rhinovirus amplicon comprises a nucleic acidsequences that contains at least one 5-Me-dC, at least one non-WatsonCrick base, at least one degenerate base, or a combination thereof, orboth.

15. The composition or kit of claim 4 or claim 5, wherein the secondtarget nucleic acid is an Rhinovirus target nucleic acid and wherein thefirst amplification oligomer configured to amplify an Rhinovirusamplicon comprises a nucleic acid sequences that contains at least one5-Me-dC, at least one non-Watson Crick base, at least one degeneratebase, or a combination thereof, or wherein the second amplificationoligomer configured to amplify an Rhinovirus amplicon comprises anucleic acid sequences that contains at least one 5-Me-dC, at least onenon-Watson Crick base, at least one degenerate base, or a combinationthereof, or both.

16. The composition or kit of any one of claim 1, 4, 7, 8, 10 or 11,wherein for the Adenovirus target nucleic acid the first amplificationoligomer comprises a target hybridizing sequence selected from the groupconsisting of SEQ ID NOS: 1, 5, 11, 12, 25, 26, 31, 32, 33, 34, 35, 38,71, 72, 73, 74.

17. The composition or kit of claim 5, 6 or 9, wherein the second targetnucleic acid is Adenovirus and wherein the first amplification oligomercomprises a target hybridizing sequence selected from the groupconsisting of SEQ ID NOS: 1, 5, 11, 12, 25, 26, 31, 32, 33, 34, 35, 38,71, 72, 73, 74.

18. The composition or kit of any one of claim 1, 4, 7, 8, 10 or 11,wherein for the Adenovirus target nucleic acid the second amplificationoligomer comprises a target hybridizing sequence selected from the groupconsisting of SEQ ID NOS: 2, 3, 6, 7, 8, 9, 13, 14, 15, 16, 27, 28, 42,43, 44, 45, 46, 61, 62, 138, 139, 140, 141, 142, 143, 144, 145, 146,147, 148, 149.

19. The composition or kit of claim 5, 6 or 9, wherein the second targetnucleic acid is Adenovirus and wherein the second amplification oligomercomprises a target hybridizing sequence selected from the groupconsisting of SEQ ID NOS: 2, 3, 6, 7, 8, 9, 13, 14, 15, 16, 27, 28, 42,43, 44, 45, 46, 61, 62, 138, 139, 140, 141, 142, 143, 144, 145, 146,147, 148, 149.

20. The composition or kit of any one of claim 2, 5, 7, 8, 12 or 13,wherein for the Metapneumovirus target nucleic acid the firstamplification oligomer comprises, consists of consists essentially of atarget hybridizing sequence selected from the group consisting of SEQ IDNOS:52, 53, 151, 152, 153, 154, 160.

21. The composition or kit of claim 4, 6, or 9, wherein the secondtarget nucleic acid is Metapneumovirus and wherein the firstamplification oligomer comprises, consists of consists essentially of atarget hybridizing sequence selected from the group consisting of SEQ IDNOS:52, 53, 151, 152, 153, 154, 160.

22. The composition or kit of any one of claim 2, 5, 7, 8, 12 or 13,wherein for the Metapneumovirus target nucleic acid the secondamplification oligomer comprises, consists of consists essentially of atarget hybridizing sequence selected from the group consisting of SEQ IDNOS:56, 68, 158, 177, 178.

23. The composition or kit of claim 4, 6, or 9, wherein the secondtarget nucleic acid is Metapneumovirus and wherein the secondamplification oligomer comprises, consists of consists essentially of atarget hybridizing sequence selected from the group consisting of SEQ IDNOS:56, 68, 158, 177, 178.

24. The composition or kit of any one of claim 3, 6, 7, 8, 14 or 15,wherein for the Rhinovirus target nucleic acid the first amplificationoligomer comprises, consists of consists essentially of a targethybridizing sequence selected from the group consisting of SEQ IDNOS:50, 51, 59, 60, 65, 75, 77 to 86, 102 to 108, 121 to 130. 25. Thecomposition or kit of claim 4, 5, or 9, wherein the second targetnucleic acid is Rhinovirus and wherein the first amplification oligomercomprises, consists of consists essentially of a target hybridizingsequence selected from the group consisting of SEQ ID NOS:50, 51, 59,60, 65, 75, 77 to 86, 102 to 108, 121 to 130.

26. The composition or kit of any one of claim 3, 6, 7, 8, 14 or 15,wherein for the Rhinovirus target nucleic acid the second amplificationoligomer comprises, consists of consists essentially of a targethybridizing sequence selected from the group consisting of SEQ IDNOS:57, 95 to 100, 115 to 119, 137.

27. The composition or kit of claim 4, 5, or 9, wherein the secondtarget nucleic acid is Rhinovirus and wherein the second amplificationoligomer comprises, consists of consists essentially of a targethybridizing sequence selected from the group consisting of SEQ IDNOS:57, 95 to 100, 115 to 119, 137.

28. The composition or kit of any one of claims 1 to 27, wherein thecomposition or kit further comprises a least one detection probeoligomer.

29. The composition or kit of any one of claims 1, 4, 7, 8 to 11, and 16to 19, wherein the composition or kit further comprises an Adenovirusdetection probe oligomer comprising a sequence selected from the groupconsisting of SEQ ID NOS:4, 10, 17, 18, 19, 20, 21, 22, 23, 24, 29, 30,36, 37, 39, 40, 63, 64, 139, 140.

30. The composition or kit of any one of claim 5 or 6, wherein thesecond target nucleic acid is Adenovirus and wherein the composition orkit further comprises a detection probe oligomer comprising a sequenceselected from the group consisting of SEQ ID Nos. 4, 10, 17, 18, 19, 20,21, 22, 23, 24, 29, 30, 36, 37, 39, 40, 63, 64, 139, 140.

31. The composition or kit of any one of claims 1, 4, 7, 8 to 11, and 16to 19, wherein the composition or kit further comprises an Adenovirusdetection probe oligomer comprising a sequence that is from 18 to 36nucleobases in length wherein the 18 to 36 nucleobases are all selectedfrom contiguous nucleobases within SEQ ID NO:138.

32. The composition or kit of any one of claim 15 or 6, wherein thesecond target nucleic acid is Adenovirus and wherein the composition orkit further comprises a detection probe oligomer comprising a sequencethat is from 18 to 36 nucleobases in length wherein the 18 to 36nucleobases are all selected from contiguous nucleobases within SEQ IDNO:138.

33. The composition or kit of any one of claims 2, 5, 7, 8, 9, 12, 13,20, 21, 22, and 23, wherein the composition or kit further comprises aMetapneumovirus detection probe oligomer comprising a sequence selectedfrom the group consisting of SEQ ID Nos. 67, 69, 70, 155, 156, 157, 161,162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175,and 176.

34. The composition or kit of any one of claim 4 or 6, wherein thesecond target nucleic acid is Metapneumovirus and wherein thecomposition or kit further comprises a detection probe oligomercomprising a sequence selected from the group consisting of SEQ ID Nos.67, 69, 70, 155, 156, 157, 161, 162, 163, 164, 165, 166, 167, 168, 169,170, 171, 172, 173, 174, 175, and 176.

35. The composition or kit of any one of claims 2, 5, 7, 8, 9, 12, 13,20, 21, 22, and 23, wherein the composition or kit further comprises aMetapneumovirus detection probe oligomer comprising a sequence that isfrom 18 to 36 nucleobases in length wherein the 18 to 36 nucleobases areall selected from contiguous nucleobases within SEQ ID NO:161 or withinSEQ ID NO:155.

36. The composition or kit of any one of claim 4 or 6, wherein thesecond target nucleic acid is Metapneumovirus and wherein thecomposition or kit further comprises a detection probe oligomercomprising a sequence that is from 18 to 36 nucleobases in lengthwherein the 18 to 36 nucleobases are all selected from contiguousnucleobases within SEQ ID NO:161 or within SEQ ID NO:155.

37. The composition or kit of any one of claims 3, 6, 7, 8, 9, 14, 15,and 24 to 27, wherein the composition or kit further comprises aRhinovirus detection probe oligomer comprising a sequence selected fromthe group consisting of SEQ ID Nos. 48, 49, 54, 87 to 94, 109 to 114,and 131 to 136. 38. The composition or kit of any one of claim 4 or 5,wherein the second target nucleic acid is Rhinovirus and wherein thecomposition or kit further comprises a detection probe oligomercomprising a sequence selected from the group consisting of SEQ ID Nos.48, 49, 54, 87 to 94, 109 to 114, 131 to 136.

39. The composition or kit of any one of claims 28 to 38, wherein atleast one of the detection probe oligomers comprises at least one5-Me-dC, or at least one non-Watson Crick base, or at least one of adegenerate base, or a combination thereof.

40. The composition or kit of any one of claims 28 to 39, wherein atleast one of the detection probe oligomers comprises a detectable label.

41. The composition or kit of claim 40, wherein the detectable label isa fluorophore.

42. The composition or kit of claim 40 or claim 41, wherein thedetection probe oligomer is a dual labeled detection probe oligomer.

43. The composition or kit of claim 42, wherein the detection probeoligomer comprises a fluorescent detectable label and a quencher moietythat can quench a fluorescent emission from the fluorescent label.

44. The composition or kit of any one of claims 1, 4, 7 to 11, 16 to 19,28 to 32, and 39 to 43, wherein the composition or kit further comprisesone or more additional amplification oligomers each of which isconfigured to amplify an Adenovirus target nucleic acid.

45. The composition or kit of claim 5 or claim 6, wherein the secondtarget nucleic acid is Adenovirus and wherein the composition or kitfurther comprises one or more additional amplification oligomers each ofwhich is configured to amplify an Adenovirus target nucleic acid.

46. The composition or kit of claim 44 or 45, wherein each of the one ormore additional amplification oligomers comprise a target hybridizingsequence separately selected from the group consisting of SEQ ID NOS: 1to 9, 11 to 16, 25 to 28, 31 to 35, 38, 42 to 46, 61, 62, and 71 to 74.

47. The composition or kit of any one of claims 2, 5, 7, 8, 9, 12, 13,20 to 23, 28, 33 to 36, and 39 to 43, wherein the composition or kitfurther comprises one or more additional amplification oligomers each ofwhich is configured to amplify a Metapneumovirus target nucleic acid.

48. The composition or kit of claim 4 or claim 6, wherein the secondtarget nucleic acid is Metapneumovirus and wherein the composition orkit further comprises one or more additional amplification oligomerseach of which is configured to amplify a Metapneumovirus target nucleicacid.

49. The composition or kit of claim 47 or 48, wherein each of the one ormore additional amplification oligomers comprise a target hybridizingsequence separately selected from the group consisting of SEQ ID NOS:52,53, 56, 68, 151, 152, 153, 154, 158, 160, 177, 178.

50. The composition or kit of any one of claims 3, 6, 7, 8, 9, 14, 15,24 to 28, and 37 to 43, wherein the composition or kit further comprisesone or more additional amplification oligomers each of which isconfigured to amplify a Rhinovirus target nucleic acid.

51. The composition or kit of claim 4 or claim 5, wherein the secondtarget nucleic acid is Rhinovirus and wherein the composition or kitfurther comprises one or more additional amplification oligomers each ofwhich is configured to amplify a Rhinovirus target nucleic acid.

52. The composition or kit of claim 47 or 48, wherein each of the one ormore additional amplification oligomers comprise a target hybridizingsequence separately selected from the group consisting of SEQ ID NOS:50,51, 57, 59, 60, 65, 75, 77 to 86, 95 to 100, 102 to 108, 115 to 119, 121to 130, 137.

53. The composition or kit of any one of claims 1, 4, 7 to 11, 16 to 19,28 to 32, and 39 to 47, wherein the composition or kit comprises atleast first and second amplification oligomers and one or moreadditional amplification oligomers configured to amplify an Adenovirustarget nucleic acid each of the amplification oligomers independentlycomprising a sequences selected from the group consisting of: SEQ IDNOS:61, 62, 71, 72, 73, and 74.

54. The composition or kit of any one of claims 2, 5, 7, 8, 9, 12, 13,20 to 23, 28, 33 to 36, 39 to 43, 46 to 49, and 53, wherein thecomposition or kit comprises at least first and second amplificationoligomers and one or more additional amplification oligomers configuredto amplify a Metapneumovirus target nucleic acid each of theamplification oligomers each independently comprising a sequencesselected from the group consisting of: SEQ ID NOS:52, 53, 56, and 58.

55. The composition or kit of any one of claims 3, 6, 7, 8, 9, 14, 15,24 to 28, 37 to 43, and 50 to 54, wherein the composition or kitcomprises at least first and second amplification oligomers and one ormore additional amplification oligomers configured to amplify aRhinovirus target nucleic acid each of the amplification oligomers eachindependently comprising a sequences selected from the group consistingof: SEQ ID NOS:50, 51, 57, 59, 60, and 65.

56. The composition or kit of any one of claims 1, 4, 7 to 11, 16 to 19,28 to 32, 39 to 47 and 53 to 55, wherein the composition or kit furthercomprises two Adenovirus detection probe oligomers, each of thedetection probe oligomers independently comprising a sequence selectedfrom the group consisting of SEQ ID NOS:63 and 64.

57. The composition or kit of any one of claims 2, 5, 7, 8, 9, 12, 13,20 to 23, 28, 33 to 36, 39 to 43, 46 to 49, and 53 to 56, wherein thecomposition or kit further comprises three Metapneumovirus detectionprobe oligomers, each of the detection probe oligomers independentlycomprising a sequence selected from the group consisting of SEQ IDNOS:67, 69 and 70.

58. The composition or kit of any one of claims 3, 6, 7, 8, 9, 14, 15,24 to 28, 37 to 43 and 50 to 57, wherein the composition or kit furthercomprises three Rhinovirus detection probe oligomers, each of thedetection probe oligomers independently comprising a sequence selectedfrom the group consisting of SEQ ID NOS:48, 49 and 54.

59. The composition or kit of any one of the preceding claims, whereinthe composition or kit further comprises a nucleic acid target captureprobe comprising a target hybridizing sequence and an immobilized probebinding region.

60. The composition or kit of claim 59, wherein the target hybridizingsequence is a poly-K nucleotide sequence.

61. The composition or kit of claim 60, wherein the poly-K nucleotidesequence is a random poly-GU sequence.

62. The composition or kit of claim 59, 60 or 61, wherein immobilizedprobe binding region is a homopolymeric nucleotide sequence, preferablycomprising a nucleotide sequence selected from the group consisting ofT₀₋₄A₁₀₋₃₆.

63. The composition or kit of any one of claims 1 to 62, wherein thecomposition further comprises an enzyme, a buffer, dNTPs, or acombination thereof.

64. A method for the determining the presence of absence of anAdenovirus target nucleic acid, a Metapneumovirus target nucleic acid, aRhinovirus target nucleic acid, or a combination thereof in a sample,the method comprising the steps of:

-   -   (A) contacting a sample with a combination of amplification        oligomers from any one of claims 1 to 58;    -   (B) performing an in vitro nucleic acid amplification reaction        wherein any of an Adenovirus target nucleic acid, a        Metapneumovirus target nucleic acid, or a Rhinovirus target        nucleic acid in the sample is used by the combination of        amplification oligomers configured to amplify that target        nucleic acid to generate an amplification product; and    -   (C) detecting the amplification product;        thereby determining the presence or absence of the target        nucleic acid in the sample.

65. The method of claim 64, wherein the sample is a sample derived froma human.

66. The method of claim 65, wherein the sample is a mucosal sample.

67. The method of claim 65 or claim 66, wherein the sample is obtainedusing a nasopharyngeal swab.

68. The method of any one of claims 64 to 67, wherein, before step (A) asample preparation step is performed to separate any target nucleic acidin the sample away from other sample components.

69. The method of claim 68, wherein the sample preparation stepcomprises a target capture step.

70. The method of claim 69, wherein the target capture step comprisecontacting the sample with a nucleic acid target capture probecomprising a target hybridizing sequence and an immobilized probebinding region.

71. The method of claim 70, wherein the target hybridizing sequence is apoly-K nucleotide sequence.

72. The method of claim 71, wherein the poly-K nucleotide sequence is arandom poly-GU sequence.

73. The method of claim 70, 71 or 72, wherein immobilized probe bindingregion is a homopolymeric nucleotide sequence, preferably comprising anucleotide sequence selected from the group consisting of T₀₋₄A₁₀₋₃₆.

74. The method of any one of claims 64 to 73, wherein the detecting step(C) is performed using one or more detection probe oligomers.

75. The method of claim 74, wherein each of the one or more detectionprobe oligomers are individually selected from the group consisting of:SEQ ID NOS:4, 10, 17, 18, 19, 20, 21, 22, 23, 24, 29, 30, 36, 37, 39,40, 63, 64, 139, 140, 67, 69, 70, 155, 156, 157, 161, 162, 163, 164,165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 48, 49, 54,87 to 94, 109 to 114, and 131 to 136.

76. The method of claim 74 or 75, wherein at least one of the detectionprobe oligomers comprises at least one 5-Me-dC, or at least onenon-Watson Crick base, or at least one of a degenerate base, or acombination thereof.

77. The method of claim 74, 75 or 76, wherein at least one of thedetection probe oligomers comprises a detectable label.

78. The method of claim 77, wherein the detectable label is afluorophore.

79. The method of claim 77 or claim 78, wherein the detection probeoligomer is a dual labeled detection probe oligomer.

80. The method of claim 79, wherein the detection probe oligomercomprises a fluorescent detectable label and a quencher moiety that canquench a fluorescent emission from the fluorescent label.

81. The method of any one of claims 64 to 80, wherein the in vitronucleic acid amplification reaction comprises thermal cycling.

82. The method of any one of claims 64 to 81, wherein the in vitronucleic acid amplification reaction comprises PCR with a polymeraseenzyme having 5′ to 3′ exonuclease activity.

83. The method of any one of claims 75 to 80, wherein the in vitronucleic acid amplification reaction is performed using an enzyme having5′ to 3′ exonuclease activity.

84. The method of any one of claims 77 to 80, wherein the in vitronucleic acid amplification reaction is performed using an enzyme having5′ to 3′ exonuclease activity and wherein an amplification product isdetected by determining a fluorescence value that is above apredetermined threshold value.

85. A system for performing one or more steps of the method of claim 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, or 84.

86. The system of claim 85, wherein the system is an automated system.

87. The system of claim 85 or 86, wherein the system performs all of thesteps of the method.

88. A method for the in vitro detection of an Adenovirus target nucleicacid, a Metapneumovirus target nucleic acid, a Rhinovirus target nucleicacid, or a combination thereof in a sample, wherein the method comprisescontacting an Adenovirus target nucleic acid, a Metapneumovirus targetnucleic acid, and/or a Rhinovirus target nucleic acid with a detectionprobe oligomer from any of claims 29 to 43, wherein hybridizationbetween the detection probe oligomers and the target nucleic acid towhich the detection probe oligomer is configured to hybridize indicatesthe presence of that target nucleic acid.

89. The method of claim 88, wherein the method comprises contacting anamplification product from the Adenovirus target nucleic acid, theMetapneumovirus target nucleic acid, and/or the Rhinovirus targetnucleic acid with the detection probe oligomer, wherein hybridizationbetween the detection probe oligomers and the amplification product towhich the detection probe oligomer is configured to hybridize indicatesthe presence of that target nucleic acid from which the amplificationproduct was generated.

90. The method of claim 88 or 89, wherein the in vitro detectionreaction is performed using an enzyme having 5′ to 3′ exonucleaseactivity.

91. The method of any one of claims 88 to 90, wherein the in vitrodetection reaction is performed using an enzyme having 5′ to 3′exonuclease activity and wherein the target nucleic acid or theamplification product generated therefrom is detected by determining afluorescence value that is above a predetermined threshold value.

92. A system for performing the in vitro detection reaction of any oneof claims 88 to 91.

93. The system of claim 92, wherein the system is an automated system.

94. The system of claim 92 or 93, wherein the system performs all of thesteps of the method.

95. A dried composition comprising one or more of the amplificationoligomers from any one of claims 1 to 27.

96. A dried composition comprising one or more of the amplificationoligomers from any one of claims 44 to 55.

97. A dried composition comprising one or more of the detection probeoligomers from any of claims 29 to 43 or 56 to 58.

98. A dried composition comprising a combination of amplificationoligomers and/or detection probe oligomers from any one of claims 1 to58.

99. The dried composition of any one of claims 95 to 98, wherein thedried composition further comprises an enzyme, dNTPs, or both.

100. The dried composition of claim 99, wherein the enzyme having 5′ to3′ exonuclease activity.

101. The dried composition of claim 99 or claim 100, wherein the enzymeis a polymerase enzyme.

102. The dried composition of any one of claims 95 to 101, wherein thedried composition has an inorganic salt concentration of 10 mM or less.

103. The dried composition of any one of claims 95 to 102, wherein thedried composition has an inorganic salt concentration of 7 mM or less.

104. The dried composition of any one of claims 95 to 103, wherein thedried composition has an inorganic salt concentration of 5 mM or less.

105. The dried composition of any one of claims 95 to 101, wherein thedried composition has an inorganic salt concentration of between about0.5 mM to about 10 mM.

DETAILED DESCRIPTION

Nucleic acid oligomer sequences are disclosed that may serve as primersfor amplification detection of Adenovirus, Metapneumovirus, and/orRhinovirus nucleic acids. These target nucleic acids may be detected ina sample by using methods of in vitro nucleic acid amplification—such asPCR (eg. Taqman™ PCR)—or transcription-mediated amplification—such asTMA or NASBA. Probes for detection of the amplified nucleic acidsequences are also described. Detection probes hybridize specifically toat least a portion of the amplified sequence, either after completion ofor during the amplification process. Methods disclosed herein can beused to amplify and detect Adenovirus, Metapneumovirus, and/orRhinovirus nucleic acids present in samples from or derived from animalsand humans.

The disclosed nucleic acid sequences and methods are useful foramplifying and detecting Adenovirus, Metapneumovirus, and/or Rhinovirusnucleic acids from or derived from viral particles present in a samplein a relatively short time so that diagnosis can be made quickly,allowing initiation of effective treatment and limiting spread of thevirus. The methods are useful for screening for individuals who haveAdenovirus, Metapneumovirus, and/or Rhinovirus infections and areparticularly useful for screening patients who have a higher risk ofdeath or serious complications from Adenovirus, Metapneumovirus, and/orRhinovirus infections, eg., the young, elderly, or immunocompromisedindividuals. The methods are also useful for rapid screening of manysamples. The methods are useful because they minimize the risk ofexposure of laboratory personnel to the infectious agents, therebylimiting the risk of infection and spread of the virus. Thus, themethods and compositions disclosed herein respond to a need for rapid,sensitive, and specific testing of clinical samples that may containAdenovirus, Metapneumovirus, and/or Rhinovirus.

The disclosed probe sequences may be used as primers, and the disclosedprimers may be used as probes. The same is true for the disclosed probedomains and primer domains. Thus, the probe domains disclosed herein maybe used as primer domains. Likewise, primer domains disclosed herein maybe used as probe domains.

The amplification oligomers disclosed herein are further contemplated ascomponents of multiplex amplification reactions wherein severaldifferent amplicon species can be produced from an assortment (eg. twoor more, three or more, for or more, five or more, six or more, or eventen or more) of target-specific primers. For example, it is contemplatedthat more than one of the amplification systems disclosed herein can becombined to result in a multiplex assay that is both robust and broad inits capacity for target detection—such as the ability to amplify anddetect nucleic acid from at least two, at least three, at least four ormore organisms. For example, the amplification systems disclosed hereincan be combined to result in a multiplex assay for target detection of:Adenovirus target nucleic acid and at least one additional targetnucleic acid; Metapneumovirus target nucleic acid and at least oneadditional target nucleic acid; Rhinovirus target nucleic acid and atleast one additional target nucleic acid; Adenovirus target nucleic acidand Metapneumovirus target nucleic acid and at least one additionaltarget nucleic acid; Adenovirus target nucleic acid and Rhinovirustarget nucleic acids and at least one additional target nucleic acid;Rhinovirus target nucleic acid and Metapneumovirus target nucleic acidand at least one additional target nucleic acid; Adenovirus targetnucleic acid and Metapneumovirus target nucleic acids and Rhinovirustarget nucleic acid and at least one additional target nucleic acid. Themultiplex assay described herein includes providing two or moreamplification systems that each amplify and detect a different subtypeor subgroup of a species, different species of an organism, or acombination thereof.

To aid in understanding aspects of the disclosure, some terms usedherein are described in more detail. All other scientific and technicalterms used herein have the same meaning as commonly understood by thoseskilled in the relevant art, such as may be provided in Dictionary ofMicrobiology and Molecular Biology, 2nd ed. (Singleton et al., 1994,John Wiley & Sons, New York, N.Y.), The Harper Collins Dictionary ofBiology (Hale & Marham, 1991, Harper Perennial, New York, N.Y.), andreferences cited herein. Unless mentioned otherwise, the techniquesemployed or contemplated herein are standard methods well known to aperson of ordinary skill in the art of molecular biology.

Definitions

It is to be noted that the term “a” or “an” “the’ entity refers to oneor more of that entity; for example, “a nucleic acid,” is understood torepresent one or more nucleic acids. As such, the terms “a” (or “an”),“one or more,” and “at least one” can be used interchangeably herein.

Sample. A “sample” or “specimen”, including “biological” or “clinical”samples may contain or may be suspected of containing Adeno, hMPV and/orHRV organisms or components thereof, such as nucleic acids or fragmentsof nucleic acids. A sample may be a complex mixture of components.Samples include “biological samples” which include any tissue ormaterial derived from a living or dead mammal or organism, including,for example, blood, plasma, serum, blood cells, saliva, mucous andcerebrospinal fluid. Samples may also include samples of in vitro cellculture constituents including, eg., conditioned media resulting fromthe growth of cells and tissues in culture medium. The sample may betreated to physically or mechanically disrupt tissue or cell structureto release intracellular nucleic acids into a solution which may containenzymes, buffers, salts, detergents and the like, to prepare the samplefor analysis. In one step of the methods described herein, a sample isprovided that is suspected of containing at least an Adeno, hMPV and/orHRV target nucleic acid. Accordingly, this step excludes the physicalstep of obtaining the sample from a subject.

Nucleic acid. This refers to a multimeric compound comprising two ormore covalently bonded nucleosides or nucleoside analogs havingnitrogenous heterocyclic bases, or base analogs, where the nucleosidesare linked together by phosphodiester bonds or other linkages to form apolynucleotide. Nucleic acids include RNA, DNA, or chimeric DNA-RNApolymers or oligonucleotides, and analogs thereof. A nucleic acid“backbone” may be made up of a variety of linkages, including one ormore of sugar-phosphodiester linkages, peptide-nucleic acid bonds (in“peptide nucleic acids” or PNAs, see PCT No. WO 95/32305),phosphorothioate linkages, methylphosphonate linkages, or combinationsthereof. Sugar moieties of the nucleic acid may be either ribose ordeoxyribose, or similar compounds having known substitutions, e.g., 2′methoxy substitutions and 2′ halide substitutions (e.g., 2′-F).Nitrogenous bases may be conventional bases (A, G, C, T, U), analogsthereof (e.g., inosine, 5 methyl 2′ deoyxcytosine (5-Me-dC), isoguanine;The Biochemistry of the Nucleic Acids 5-36, Adams et al., ed., 11th ed.,1992, Abraham et al., 2007, BioTechniques 43: 617-24), which includederivatives of purine or pyrimidine bases (e.g., N⁴-methyldeoxygaunosine, deaza- or aza-purines, deaza- or aza-pyrimidines,pyrimidine bases having substituent groups at the 5 or 6 position,purine bases having an altered or replacement substituent at the 2, 6and/or 8 position, such as 2-amino-6-methylaminopurine,O⁶-methylguanine, 4-thio-pyrimidines, 4-amino-pyrimidines,4-dimethylhydrazine-pyrimidines, and O⁴-alkyl-pyrimidines, andpyrazolo-compounds, such as unsubstituted or 3-substitutedpyrazolo[3,4-d]pyrimidine; U.S. Pat. Nos. 5,378,825, 6,949,367 and PCTNo. WO 93/13121). Nucleic acids may include “abasic” residues in whichthe backbone does not include a nitrogenous base for one or moreresidues (U.S. Pat. No. 5,585,481). A nucleic acid may comprise onlyconventional sugars, bases, and linkages as found in RNA and DNA, or mayinclude conventional components and substitutions (e.g., conventionalbases linked by a 2′ methoxy backbone, or a nucleic acid including amixture of conventional bases and one or more base analogs). Nucleicacids may include “locked nucleic acids” (LNA), in which one or morenucleotide monomers have a bicyclic furanose unit locked in an RNAmimicking sugar conformation, which enhances hybridization affinitytoward complementary sequences in single-stranded RNA (ssRNA),single-stranded DNA (ssDNA), or double-stranded DNA (dsDNA) (Vester etal., 2004, Biochemistry 43(42):13233-41). Nucleic acids may includemodified bases to alter the function or behaviour of the nucleic acid,e.g., addition of a 3′-terminal dideoxynucleotide to block additionalnucleotides from being added to the nucleic acid. Synthetic methods formaking nucleic acids in vitro are well known in the art although nucleicacids may be purified from natural sources using routine techniques.

Polynucleotide and Oligonucleotide. These terms denote a nucleic acidchain. Throughout this application, nucleic acids are designated by the5′-terminus to the 3′-terminus. Standard nucleic acids, e.g., DNA andRNA, are typically synthesized “3′-to-5′,” i.e., by the addition ofnucleotides to the 5′-terminus of a growing nucleic acid.Oligonucleotide is may be used interchangeably with “oligomer and“oligo” and refers to a nucleic acid having generally less than 1,000nucleotide (nt) residues, including polymers in a range of from about 5nt residues to about 900 nt residues, from about 10 nt residues to about800 nt residues with a lower limit of about 12 to 15 nt and an upperlimit of about 40 to 600 nt, and other embodiments are in a range havinga lower limit of about 15 to 20 nt and an upper limit of about 22 to 100nt. It is understood that these ranges are exemplary only, and anoligonucleotide may contain each whole number included in the range.Oligonucleotides may be purified from naturally occurring sources, butmay be synthesized using any of a variety of well-known enzymatic orchemical methods. The term oligonucleotide does not denote anyparticular function to the reagent; rather, it is used generically tocover all such reagents described herein. An oligonucleotide may servevarious different functions. For example, it may function as a primer ifit is specific for and capable of hybridizing to a complementary strandand can further be extended in the presence of a nucleic acidpolymerase, it may provide a promoter if it contains a sequencerecognized by an RNA polymerase and allows for transcription (eg., a T7provider), and it may function to prevent hybridization or impede primerextension if appropriately situated and/or modified.

Nucleotide. This is a subunit of a nucleic acid consisting of aphosphate group, a 5-carbon sugar and a nitrogenous base. The 5-carbonsugar found in RNA is ribose. In DNA, the 5-carbon sugar is2′-deoxyribose. The term also includes analogs of such subunits, such asa methoxy group at the 2′ position of the ribose (2′-O-Me, or 2′methoxy).

As used herein, methoxy oligonucleotides containing “T” residues have amethoxy group at the 2′ position of the ribose moiety, and a uracil atthe base position of the nucleotide. “C residues” present in anoligonucleotide (e.g., a primer or probe) include methylated (e.g.,5-Me-dC) and unmethylated cytosines unless the context indicatesotherwise.

Non-nucleotide unit. This is a unit that does not significantlyparticipate in hybridization of a polymer. Such units must not, forexample, participate in any significant hydrogen bonding with anucleotide, and would exclude units having as a component one of thefive nucleotide bases or analogs thereof.

Target nucleic acid. This is a nucleic acid comprising a “targetsequence” to be amplified. Target nucleic acids may be DNA or RNA andmay be either single-stranded or double-stranded. The target nucleicacid may include other sequences besides the target sequence that may beamplified. Typical target nucleic acids are or are derived from the Adv,hMPV and HRV genomes.

Target sequence. This term refers to the particular nucleotide sequenceof the target nucleic acid that is to be amplified. Where the targetnucleic acid is originally single-stranded, the term “target sequence”will also refer to the sequence complementary to the target sequence aspresent in the target nucleic acid. Where the target nucleic acid isoriginally double-stranded, the term “target sequence” refers to boththe sense (+) and antisense (−) strands. The terms “target(s) asequence” or “target(s) a target nucleic acid” as used herein inreference to a region of an Adeno, hMPV or HRV nucleic acid refers to aprocess whereby an oligonucleotide stably hybridizes to the targetsequence in a manner that allows for amplification and/or detection asdescribed herein. In one embodiment, the oligonucleotide iscomplementary to the target sequence and contains no mismatches. Inanother embodiment, the oligonucleotide is complementary but contains 1;or 2; or 3; or 4; or 5 or more mismatches with the target sequence. Inanother embodiment, the oligonucleotide is complementary to the targetsequence but contains one or a combination of a degenerate nucleotideresidue, a non-Watson Crick residue, or a nucleoside analog. Preferably,the oligonucleotide that stably hybridizes to the target sequenceincludes at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 30, 35, 40, 45 or 50 contiguous nucleotides complementary tothe target sequence. It is understood that at least 10 and as many as 50is an inclusive range such that 10, 50 and each whole number therebetween are included. The term “configured to target a sequence” as usedherein means that the target hybridizing region of an amplificationoligonucleotide is designed to have a polynucleotide sequence that couldtarget a sequence of the referenced Adeno, hMPV or HRV region. Such anamplification oligonucleotide is not limited to targeting that sequenceonly, but is rather useful as a composition, in a kit or in a method fortargeting an Adeno, hMPV or HRV target nucleic acid, as is describedherein. The term “configured to” denotes an actual arrangement of thepolynucleotide sequence configuration of the amplificationoligonucleotide target hybridizing sequence.

Isolated, Separating, Purifying. refer to taking a nucleic acid from itsnatural milieu, but these terms do not necessarily connote any degree ofpurification. These terms mean that one or more components of a sampleare removed or separated from other sample components. Sample componentsinclude target nucleic acids usually in a generally aqueous solutionphase, which may also include cellular fragments, proteins,carbohydrates, lipids, and other nucleic acids. Separating or purifyingremoves at least 70%, or at least 80%, or at least 95% of the targetnucleic acid from other sample components. Ranges of %-purity includeall whole and rational numbers of the range.

Region. This term refers to a portion of a nucleic acid wherein saidportion is smaller than the entire nucleic acid. For example, when thenucleic acid in reference is an oligonucleotide promoter provider, theterm “region” may be used refer to the smaller promoter portion of theentire oligonucleotide. Similarly, and also as example only, when thenucleic acid is a target nucleic acid, the term “region” may be used torefer to a smaller area of the nucleic acid.

By “RNA and DNA equivalents” is meant RNA and DNA molecules havingessentially the same complementary base pair hybridization properties.RNA and DNA equivalents have different sugar moieties (i.e., riboseversus deoxyribose) and may differ by the presence of uracil in RNA andthymine in DNA. The differences between RNA and DNA equivalents do notcontribute to differences in homology because the equivalents have thesame degree of complementarity to a particular sequence. Unlessotherwise indicated, reference to an Adv, hMPV or HRV nucleic acidincludes the respective Adv, hMPV, or HRV RNA and DNA equivalentsthereof.

As used herein, an oligonucleotide having a nucleic acid sequence“comprising” or “consisting of” or “consisting essentially of” asequence selected from a group of specific sequences means that theoligonucleotide, as a basic and novel characteristic, is capable ofstably hybridizing to a nucleic acid having the exact complement of oneof the listed nucleic acid sequences of the group under stringenthybridization conditions. An exact complement includes the correspondingDNA or RNA sequence.

Corresponds. As used herein, a nucleic acid “corresponds” to a specifiednucleic acid if the nucleic acid is 100% identical or complementary tothe specified nucleic acid.

Substantially corresponding to. As used herein, a nucleic acid“substantially corresponding to” a specified nucleic acid sequence meansthat the referred to oligonucleotide is sufficiently similar to thereference nucleic acid sequence such that the oligonucleotide hassimilar hybridization properties to the reference nucleic acid sequencein that it would hybridize with the same target nucleic acid sequenceunder stringent hybridization conditions. Substantially correspondingnucleic acids vary by at least one nucleotide from the specified nucleicacid. This variation may be stated in terms of a percentage of identityor complementarity between the nucleic acid and the specified nucleicacid. Thus, nucleic acid substantially corresponds to a referencenucleic acid sequence if these percentages of base identity orcomplementarity are from less than 100% to about 80% (inclusive of allwhole and rational numbers therein).

Blocking moiety. As used herein, a “blocking moiety” is a substance usedto “block” the 3′-terminus of an oligonucleotide or other nucleic acidso that it cannot be efficiently extended by a nucleic acid polymerase.Oligomers not intended for primer-based extension by a nucleic acidpolymerase may include a blocker group that replaces the 3′OH to preventthe enzyme-mediated extension of the oligomer in an amplificationreaction. For example, blocked amplification oligomers and/or detectionprobes present during amplification may not have functional 3′OH andinstead include one or more blocking groups located at or near the 3′end. In some embodiments a blocking group near the 3′ end and may bewithin five residues of the 3′ end and is sufficiently large to limitbinding of a polymerase to the oligomer. In other embodiments a blockinggroup is covalently attached to the 3′ terminus. Many different chemicalgroups may be used to block the 3′ end, e.g., alkyl groups,non-nucleotide linkers, alkane-diol dideoxynucleotide residues, andcordycepin.

Amplification oligomer. An “amplification oligomer”, which may also becalled an “amplification oligonucleotide” or a “primer” is an oligomer,at least the 3′-end of which is complementary to a target nucleic acid(“target hybridizing sequence”), and which hybridizes to a targetnucleic acid, or its complement, and participates in a nucleic acidamplification reaction. An example of an amplification oligomer is aprimer that hybridizes to a target nucleic acid and contains a 3′ OH endthat is extended by a polymerase in an amplification process. Anotherexample of an amplification oligomer is a “promoter-based amplificationoligomer,” which comprises a target hybridizing sequence, and a promotersequence for initiating transcription by an appropriate polymerase. Sizeranges for amplification oligonucleotides include those comprisingtarget hybridizing regions that are about 10 to about 70 nt long(inclusive of all whole and rational numbers therein). In oneembodiment, an amplification oligomer may optionally contains one or acombination of a degenerate nucleotide residue, a non-Watson Crickresidue, or a nucleoside analog. An amplification oligomer designated tocomprise at least one degenerate nucleobase is thus a collection ofamplification oligomer species each independently having one of thenucleic acid residues represented by the degenerate nucleotide.

Amplification. This refers to any known procedure for obtaining multiplecopies of a target nucleic acid sequence or its complement or fragmentsthereof. The multiple copies may be referred to as amplicons oramplification products. Amplification of “fragments” refers toproduction of an amplified nucleic acid that contains less than thecomplete target nucleic acid or its complement, eg., produced by usingan amplification oligonucleotide that hybridizes to, and initiatespolymerization from, an internal position of the target nucleic acid.Known amplification methods include both thermally cyclical andisothermal amplification methods. For some embodiment, isothermalamplification methods are preferred. Replicase-mediated amplification,polymerase chain reaction (PCR), ligase chain reaction (LCR),strand-displacement amplification (SDA), and transcription-mediated ortranscription-associated amplification are non-limiting examples ofnucleic acid amplification methods (see e.g., U.S. Pat. Nos. 4,786,600;4,683,195, 4,683,202, and 4,800,159; 5,427,930 and 5,516,663; and5,422,252; 5,547,861; and 5,648,211).

By “assay conditions” is meant conditions permitting stablehybridization of an oligonucleotide to a target nucleic acid. Assayconditions do not require preferential hybridization of theoligonucleotide to the target nucleic acid.

In cyclic amplification methods that detect amplicons in real-time, theterm “Threshold cycle” (Ct) is a measure of the emergence time of asignal associated with amplification of target, and is generally 10×standard deviation of the normalized reporter signal. Once anamplification reaches the “threshold cycle,” generally there isconsidered to be a positive amplification product of a sequence to whichthe probe binds. The identity of the amplification product can then bedetermined through methods known to one of skill in the art, such as gelelectrophoresis, nucleic acid sequencing, and other such well knownmethods.

Real-time amplification. As used herein, the term “real-timeamplification” refers to amplification of target nucleic acid that ismonitored by real-time detection means.

Amplicon. This term, which is used interchangeably with “amplificationproduct”, refers to the nucleic acid molecule generated during anamplification procedure that is complementary or homologous to asequence contained within the target sequence. These terms can be usedto refer to a single strand amplification product, a double strandamplification product or one of the strands of a double strandamplification product.

Probe. A probe, also known as a “detection probe” or “detectionoligonucleotide” are terms referring to a nucleic acid oligomer thathybridizes specifically to a target sequence in a nucleic acid, or in anamplified nucleic acid, under conditions that promote hybridization toallow detection of the target sequence or amplified nucleic acid.Detection may either be direct (e.g., a probe hybridized directly to itstarget sequence) or indirect (e.g., a probe linked to its target via anintermediate molecular structure). Probes may be DNA, RNA, analogsthereof or combinations thereof and they may be labeled or unlabeled. Aprobe's “target sequence” generally refers to a smaller nucleic acidsequence within a larger nucleic acid sequence that hybridizesspecifically to at least a portion of a probe oligomer by standard basepairing. A probe may comprise target-specific sequences and othersequences that contribute to the three-dimensional conformation of theprobe (eg., U.S. Pat. Nos. 5,118,801; 5,312,728; 6,849,412; 6,835,542;6,534,274; and 6,361,945; and US Pub. No. 20060068417). In a preferredembodiment, the detection probe comprises a 2′ methoxy backbone whichcan result in a higher signal being obtained. In another preferredembodiment, the probe comprises a fluorophore covalently attached to the5′-end of the probe and a quencher at the 3′-end. Such probes are knownas Taqman™ probes. In another embodiment, a probe may optionallycontains one or a combination of a degenerate nucleotide residue, anon-Watson Crick residue, or a nucleoside analog. A probe designated tocomprise at least one degenerate nucleobase is thus a collection ofprobe species each independently having one of the nucleic acid residuesrepresented by the degenerate nucleotide.

Stable. By “stable” or “stable for detection” is meant that thetemperature of a reaction mixture is at least 2.deg.C. below the meltingtemperature of a nucleic acid duplex.

Label. As used herein, a “label” refers to a moiety or compound joineddirectly or indirectly to a probe that is detected or leads to adetectable signal. Direct labeling can occur through bonds orinteractions that link the label to the probe, including covalent bondsor non-covalent interactions, e.g. hydrogen bonds, hydrophobic and ionicinteractions, or formation of chelates or coordination complexes.Indirect labeling can occur through use of a bridging moiety or “linker”such as a binding pair member, an antibody or additional oligomer, whichis either directly or indirectly labeled, and which may amplify thedetectable signal. Labels include any detectable moiety, such as aradionuclide, ligand (e.g., biotin, avidin), enzyme or enzyme substrate,reactive group, or chromophore (e.g., dye, particle, or bead thatimparts detectable color), luminescent compound (e.g., bioluminescent,phosphorescent, or chemiluminescent labels (e.g., U.S. Pat. Nos.5,656,207, 5,658,737, and 5,639,604)), or fluorophore. Labels may bedetectable in a homogeneous assay in which bound labeled probe in amixture exhibits a detectable change different from that of an unboundlabeled probe, e.g., instability or differential degradation properties(e.g., U.S. Pat. Nos. 5,283,174, 5,656,207, and 5,658,737). Embodimentsof fluorophores include those that absorb light in the range of about495 to 650 nm and emit light in the range of about 520 to 670 nm, whichinclude those known as FAM™, TET™, CAL FLUOR™ (Orange or Red), andQUASAR™ compounds. Fluorophores may be used in combination with aquencher molecule that absorbs light when in close proximity to thefluorophore to diminish background fluorescence. Such quenchers are wellknown in the art and include, e.g., BLACK HOLE QUENCHER™ (or BHQ™,BHQ-1™, or BHQ-2™) or TAMRA™ compounds. Synthesis and methods ofattaching labels to nucleic acids and detecting labels are well known(e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed.(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),Chapter 10; U.S. Pat. Nos. 5,658,737, 5,656,207, 5,547,842, 5,283,174,and 4,581,333). More than one label, and more than one type of label,may be present on a particular probe, or detection may use a mixture ofprobes in which each probe is labeled with a compound that produces adifferent detectable signal (e.g., U.S. Pat. Nos. 6,180,340 and6,350,579).

Capture oligonucleotide. As used herein, a “capture oligonucleotide,”“target capture oligonucleotide” or “capture probe” refers to a nucleicacid oligomer that hybridizes to a target sequence in a target nucleicacid by and joins to a binding partner on an immobilized probe tocapture the target nucleic acid to a support. One example of a captureoligomer includes an oligonucleotide comprising two binding regions: atarget hybridizing sequence and an immobilized probe-binding region. Avariation of this example, the two regions may be present on twodifferent oligomers joined together by one or more linkers. Anotherembodiment of a capture oligomer the target hybridizing sequence is asequence that includes random or non-random poly-K poly-GU, poly-GT, orpoly U sequences to bind non-specifically to a target nucleic acid andlink it to an immobilized probe on a support. (see e.g., PCT Pub No. WO2008/016988 and U.S. Pat. No. 9,051,601). The immobilized probe bindingregion can be a nucleic acid sequence, referred to as a tail. Tailsinclude a substantially homopolymeric tail of about 10 to 40 nucleotides(e.g., A₁₀ to A₄₀), or of about 14 to 33 nt (e.g., T₃A₁₄ to T₃A₃₀), thatbind to a complementary immobilized sequence. Thus, a non-limitingexample of preferred nucleic acid tails can in some embodiments includeT₀₋₄A₁₀₋₃₆ sequences.

Immobilized oligonucleotide. As used herein, an “immobilizedoligonucleotide”, “immobilized probe” or “immobilized nucleic acid”refers to a nucleic acid binding partner that joins a capture oligomerto a support, directly or indirectly to facilitate separation of acapture probe bound target nucleic acid from unbound material in asample. One embodiment of an immobilized probe is an oligomer joined toa support, such as nitrocellulose, nylon, glass, polyacrylate, mixedpolymers, polystyrene, silane, polypropylene, metal, or othercompositions, of which one embodiment is magnetically attractableparticles.

Complementary. By “complementary” is meant that the nucleotide sequencesof similar regions of two single-stranded nucleic acids, or to differentregions of the same single-stranded nucleic acid have a nucleotide basecomposition that allow the single-stranded regions to hybridize togetherin a stable double-stranded hydrogen-bonded region under stringenthybridization or amplification conditions. Sequences that hybridize toeach other may be completely complementary or partially complementary tothe intended target sequence by standard nucleic acid base pairing (e.g.G:C, A:T or A:U pairing). By “sufficiently complementary” is meant acontiguous sequence that is capable of hybridizing to another sequenceby hydrogen bonding between a series of complementary bases, which maybe complementary at each position in the sequence by standard basepairing or may contain one or more residues that are not complementaryby standard A:T/U and G:C pairing, or are modified nucleotides such asabasic residues, modified nucleotides or nucleotide analogs.Sufficiently complementary contiguous sequences typically are at least80%, or at least 90%, complementary to a sequence to which an oligomeris intended to specifically hybridize (a %-complementarity rangeincludes all whole and rational numbers of the range). Sequences thatare “sufficiently complementary” allow stable hybridization of a nucleicacid oligomer with its target sequence under appropriate hybridizationconditions, even if the sequences are not completely complementary. Whena contiguous sequence of nucleotides of one single-stranded region isable to form a series of “canonical” hydrogen-bonded base pairs with ananalogous sequence of nucleotides of the other single-stranded region,such that A is paired with U or T and C is paired with G, thenucleotides sequences are “completely” complementary.

Preferentially hybridize. By “preferentially hybridize” is meant thatunder stringent hybridization assay conditions, an oligonucleotidehybridizes to its target sequences, or replicates thereof, to formstable oligonucleotide: target sequence hybrid, while at the same timeformation of stable oligonucleotide: non-target sequence hybrid isminimized. For example, a probe oligonucleotide preferentiallyhybridizes to a target sequence or replicate thereof to a sufficientlygreater extent than to a non-target sequence, to enable one havingordinary skill in the art to accurately detect the RNA replicates orcomplementary DNA (cDNA) of the target sequence formed during theamplification. Appropriate hybridization conditions are well known inthe art for probe, amplification, target capture, blocker and otheroligonucleotides, may be predicted based on sequence composition, or canbe determined by using routine testing methods (e.g., Sambrook et al.,Molecular Cloning, A Laboratory Manual, 2^(nd) ed. (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989) at §§ 1.90-1.91,7.37-7.57, 9.47-9.51 and 11.47-11.57, particularly §§ 9.50-9.51,11.12-11.13, 11.45-11.47 and 11.55-11.57).

Nucleic acid hybrid. By “nucleic acid hybrid” or “hybrid” or “duplex” ismeant a nucleic acid structure containing a double-stranded,hydrogen-bonded region wherein each strand is complementary to theother, and wherein the region is sufficiently stable under stringenthybridization conditions to be detected by means including, but notlimited to, chemiluminescent or fluorescent light detection,autoradiography, or gel electrophoresis. Such hybrids may compriseRNA:RNA, RNA:DNA, or DNA:DNA duplex molecules.

Sample preparation. This refers to any steps or methods that treat asample for subsequent amplification and/or detection of one or more ofan Adv, hMPV or HRV nucleic acid present in the sample. The targetnucleic acid may be a minority component in the sample. Samplepreparation may include any known method of isolating or concentratingcomponents, such as viruses or nucleic acids using standard microbiologymethods. Sample preparation may include physical disruption and/orchemical lysis of cellular components to release intracellularcomponents into a substantially aqueous or organic phase and removal ofdebris, such as by using filtration, centrifugation or adsorption.Sample preparation may include use of a nucleic acid oligonucleotidethat selectively or non-specifically captures a target nucleic acid andseparates it from other sample components (eg., as described in U.S.Pat. Nos. 6,110,678; 9,051,601, and PCT Pub. No. WO 2008/016988).

Specificity. The term “specificity,” in the context of an amplificationsystem, is used herein to refer to the characteristic of anamplification system which describes its ability to distinguish betweentarget and non-target sequences dependent on sequence and assayconditions. In terms of nucleic acid amplification, specificitygenerally refers to the ratio of the number of specific ampliconsproduced to the number of side-products (e.g., the signal-to-noiseratio).

Sensitivity. The term “sensitivity” is used herein to refer to theprecision with which a nucleic acid amplification reaction can bedetected or quantitated. The sensitivity of an amplification reaction isgenerally a measure of the smallest copy number of the target nucleicacid that can be reliably detected in the amplification system, and willdepend, for example, on the detection assay being employed, and thespecificity of the amplification reaction, e.g., the ratio of specificamplicons to side-products.

Relative fluorescence unit. As used herein, the term “relativefluorescence unit” (“RFU”) is an arbitrary unit of measurement offluorescence intensity. RFU varies with the characteristics of thedetection means used for the measurement.

References, particularly in the claims, to “the sequence of SEQ ID NO:X” refer to the base sequence of the corresponding sequence listingentry and do not require identity of the backbone (e.g., RNA, 2′-O-MeRNA, or DNA) or base modifications (e.g., methylation of cytosineresidues) unless otherwise indicated.

A “degenerate” position in an oligomer refers to a position where morethan one base pair is present in a population of the oligomer. Forexample, in SEQ ID NO: 81, the eighth nucleotide is R, which representsG or A. Oligomers with degenerate positions can be synthesized byproviding a mixture of nucleotide precursors corresponding to thedesired degenerate combination at the step of the synthesis whereincorporation of a degenerate position is desired. The resultingoligomers are a mixture of species each containing one of thenucleotides represented by the degenerate designation.

A “non-Watson Crick” (NWC) position in an oligomer refers to a positionwhere the oligomer is configured to hybridize to at least one targetnucleic acid sequence with a non-Watson Crick pairing, such as G-U, G-T,or G-A (either the G or the U/T/A can be the base in the oligomer). Insome embodiments, the NWC position is configured to hybridize via awobble (G-U or G-T) or purine-purine (G-A) pair.

Oligonucleotides for the Amplification of Adenovirus, Metapneumovirus,and/or Rhinovirus

Oligonucleotides for amplifying an each of an Adenovirus,Metapneumovirus, and/or Rhinovirus target nucleic acid typicallycomprise at least two amplification oligomers per target. Someembodiments of the disclosure may utilise two, three, four, five, six ormore amplification oligomers per target in, for example, multiplexamplification assays. Thus, by way of example, oligonucleotides foramplifying each target organism may comprise one, two, three, four, fiveor more forward amplification primers and one, two, three, four, five ormore reverse amplification primers. For example, oligonucleotides foramplifying Adenovirus may comprise one, two, three, four, five or moreforward amplification primers and one, two, three, four, five or morereverse amplification primers. Oligonucleotides for amplifying hMPV maycomprise one, two, three, four, five or more forward amplificationprimers and one, two, three, four, five or more reverse amplificationprimers. Oligonucleotides for amplifying HRV may comprise one, two,three, four, five or more forward amplification primers and one, two,three, four, five or more reverse amplification primers. Moreover, thesubtypes or subgroups of a target organism may require at least twoamplification oligomers, each comprising a nucleotide sequence that isspecific for a different member or members of one or more of theorganism's subtypes/subgroups.

Oligonucleotides for detecting each of an Adenovirus, Metapneumovirus,and/or Rhinovirus target nucleic acid typically comprise at least onedetection oligomer per target. Some embodiments of the disclosure mayutilise two, three, four, five, six or more detection probe oligomersper target in, for example, a multiplex detection assay. For example,oligonucleotides for detecting Adenovirus may comprise one, two, three,four or more detection probe oligomers. Oligonucleotides for detectinghMPV may comprise one, two, three, four or more detection probeoligomers. Oligonucleotides for detecting HRV may comprise one, two,three, four or more detection probe oligomers. Moreover, the subtypes orsubgroups of a target organism may require at least two detection probeoligomers, each comprising a nucleotide sequence that is specific for adifferent member or members of one or more of the organism'ssubtypes/subgroups. Combinations of oligomers for the multiplexedamplifying and detecting of one or more of Adenovirus, Metapneumovirus,and Rhinovirus target nucleic acids typically comprise at least twoforward amplification oligomers, at least two reverse amplificationoligomers, and at least two detection probe oligomers. Some embodimentsof the disclosure may utilise two, three, four, five, or even six ormore amplification oligomers and two, three, four, five or even six ormore probes for each intended target nucleic acid. Thus, by way ofexample, oligonucleotides for the multiplexed amplifying and detectingof a number of targets may comprise from 6 to 40 amplification oligomersand from 3 to 15 detection probe oligomers.

The methods for detecting an Adenovirus, Metapneumovirus, and/orRhinovirus target nucleic acid (including an amplicon) optionallyinclude a detecting step that uses at least one probe that bindsspecifically to the amplified product (RNA or DNA amplicon, preferablyDNA amplicon). Preferably, the probe is labeled and produces a signaldetected in a homogeneous system, that is, without separation of boundprobe from unbound probe. Other examples of probes may be labeled with afluorescent compound which emits a detectable signal only when the probeis bound to its target, e.g., a Taqman™ detection probe as describedherein.

In one embodiment, at least one of the amplification oligomers isconfigured to specifically hybridize to a region within a targetsequence of Adenovirus corresponding to nucleotides 1 to 99 of SEQ IDNo. 47, or corresponding to nucleotides 83 to 175 of SEQ ID No. 47. Inone embodiment, at least two amplification oligomers are used, whereineach of said at least two amplification oligomers is from 10 to about 50nucleotides in length and wherein the amplification oligomers arerespectively configured to specifically hybridize to regions within atarget sequence of Adenovirus selected from the group consisting of fromnucleotides 1 to 99 of SEQ ID No. 47 and from nucleotides 83 to 175 ofSEQ ID No. 47 in order to generate an amplicon that can be subsequentlydetected. In one embodiment, at least one of the amplification oligomersis configured to specifically hybridize to a region within a targetsequence of Adenovirus corresponding to nucleotides 52 to 99 and/or 40to 87 and/or 1 to 23 and/or 7 to 23 and/or 7 to 45 and/or 139 to 155and/or 103 to 175 and/or 83 to 99 and/or 83 to 98 SEQ ID No. 47 in orderto generate an amplicon that can be subsequently detected. In oneembodiment, at least one of the amplification oligomers is configured tospecifically hybridize to a region within a target sequence ofAdenovirus corresponding to nucleotides 52 to 74 and/or 76 to 99 and/or40 to 56 and/or 65 to 87 and/or 1 to 18 and/or 7 to 23 and/or 28 to 45and/or 27 to 45 and/or 26 to 45 and/or 139 to 155 and/or 103 to 123and/or 159 to 175 and/or 83 to 99 and/or 83 to 98 of SEQ ID No. 47 inorder to generate an amplicon that can be subsequently detected.

Oligonucleotides for amplifying and/or detecting the Adenovirus targetnucleic acid include oligonucleotide sequences selected from the groupconsisting of SEQ ID NOS: 1 to 46, 62 to 64, 71 to 75, and 138 to 149.Embodiments of amplification oligomers specific for Adenovirus nucleicacid include the amplification oligomers comprising, consisting orconsisting essentially of a target hybridizing sequence selected fromthe group consisting of SEQ ID NOS: 1 to 9, 11 to 16, 25 to 28, 31 to35, 38, 42 to 46, 61, 62, and 71 to 74 or a combination of two or morethereof. According to one embodiment, at least one first amplificationoligomer comprises, consists of consists essentially of a targethybridizing sequence selected from the group consisting of SEQ ID NOS:1, 5, 11, 12, 25, 26, 31, 32, 33, 34, 35, 38, 71, 72, 73, 74, or acombination of two or more thereof. According to one embodiment, atleast one second amplification oligomer comprises, consists of consistsessentially of a target hybridizing sequence selected from the groupconsisting of SEQ ID NOS: 2, 3, 6, 7, 8, 9, 13, 14, 15, 16, 27, 28, 42,43, 44, 45, 46, 61, 62, 138, 139, 140, 141, 142, 143, 144, 145, 146,147, 148, 149, or a combination of two or more thereof. In oneembodiment, one or more of the amplification oligomers for amplifying anAdenovirus target nucleic acid comprises at least one of a 5-Me-dC,non-Watson Crick base, degenerate base, or combination thereof.

In one embodiment, one or more detection probes are configured to detecta sequence in a region corresponding to nucleotides 74 to 139 of SEQ IDNO:47; and/or nucleotides 56 to 103 of SEQ ID NO:47; and/or nucleotides18 to 83 of SEQ ID NO:47; and/or nucleotides 23 to 83 of SEQ ID NO:47;and/or nucleotides 23 to 83 of SEQ ID NO:47; and/or nucleotides 23 to 83of SEQ ID NO:47 and/or nucleotides 52 to 99 of SEQ ID NO:47. In oneembodiment, there is provided a detection probe oligonucleotide that isfrom 18 to 36 nucleobases in length wherein the 18 to 36 nucleobases areall selected from contiguous nucleobases within SEQ ID NO:138.

Probes for the specific detection of Adenovirus sequences includeoligomers selected from the group consisting of SEQ ID Nos. 4, 10, 17,18, 19, 20, 21, 22, 23, 24, 29, 30, 36, 37, 39, 40, 63, 64, 139, 140 ora combination of two or more thereof. In one embodiment, one or more ofthe detection probe oligomers for detecting an Adenovirus target nucleicacid (including an Adenovirus amplicon) comprises at least one of a5-Me-dC, non-Watson Crick base, degenerate base, or combination thereof.

Oligonucleotides for amplifying and/or detecting an hMPV target nucleicacid include oligonucleotide sequences that are configured to hybridizeto a region of an hMPV in a region corresponding to nucleotides 966 to1147 of SEQ ID NO:150, and/or nucleotides 844 to 1027 of SEQ ID NO:159,and/or 1000 to 1040 of SEQ ID NO:150, and/or 880 to 915 of SEQ IDNO:159, and/or 1027 to 1080 of SEQ ID NO:150, and/or 913 to 958 of SEQID NO:159, and/or 1073 to 1115 of SEQ ID NO:150, and/or 953 to 995 ofSEQ ID NO:159. Oligonucleotides for amplifying and/or detecting an hMPVtarget nucleic acid include oligonucleotide sequences selected from thegroup consisting of SEQ ID NOS:52, 53, 56, 67 to 70, 151 to 158, and 161to 178. Embodiments of amplification oligomers include the amplificationoligomers comprising, consisting or consisting essentially of a targethybridizing sequence selected from the group consisting of SEQ IDNOS:52, 53, 56, 68, 151, 152, 153, 154, 158, 160, 177, 178, or acombination of two or more thereof. Embodiments of first amplificationoligomers include the amplification oligomers comprising, consisting orconsisting essentially of a target hybridizing sequence selected fromthe group consisting of SEQ ID NOS:52, 53, 151, 152, 153, 154, 160, or acombination of two or more thereof. Embodiments of second amplificationoligomers include the amplification oligomers comprising, consisting orconsisting essentially of a target hybridizing sequence selected fromthe group consisting of SEQ ID NOS:56, 68, 158, 177, 178, or acombination of two or more thereof. In one embodiment, one or more ofthe amplification oligomers for amplifying an hMPV target nucleic acidcomprises at least one of a 5-Me-dC, non-Watson Crick base, degeneratebase, or combination thereof.

Probes for the specific detection of hMPV sequences include oligomersselected from the group consisting of SEQ ID Nos. 67, 69, 70, 155, 156,157, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173,174, 175, 176, or a combination of two or more thereof. In oneembodiment, there is provided a detection probe oligonucleotide that isfrom 18 to 36 nucleobases in length wherein the 18 to 36 nucleobases areall selected from contiguous nucleobases within SEQ ID NO:161 or withinSEQ ID NO:155. In one embodiment, one or more of the detection probeoligomers for detecting an hMPV target nucleic acid (including an hMPVamplicon) comprises at least one of a 5-Me-dC, non-Watson Crick base,degenerate base, or combination thereof.

Oligonucleotides for amplifying and/or detecting an HRV target nucleicacid include oligonucleotide sequences that are configured to hybridizeto a region of an HRV in a region corresponding to nucleotides 230 to556 of SEQ ID NO:120, and/or 199 to 525 of SEQ ID NO:101, and/or 80 to410 of SEQ ID NO:76, and/or 263 to 303 of SEQ ID NO:120, and/or 231 to264 of SEQ ID NO:101, and/or 106 to 156 of SEQ ID NO:76, and/or 312 to346 of SEQ ID NO:120, and/or 279 to 314 of SEQ ID NO:101, and/or 455 to506 of SEQ ID NO:76, and/or 480 to 533 of SEQ ID NO:120, and/or 455 to506 of SEQ ID NO:101, and/or 338 to 397 of SEQ ID NO:76.Oligonucleotides for amplifying and/or detecting an HRV target nucleicacid include oligonucleotide sequences selected from the groupconsisting of SEQ ID NOS:48, 49, 50, 51, 54, 57, 59, 60, 65, 75, 77 to100, 102 to 119, 121 to 137, or a combination of two or more thereof.Embodiments of amplification oligomers include the amplificationoligomers comprising, consisting or consisting essentially of a targethybridizing sequence selected from the group consisting of SEQ IDNOS:50, 51, 57, 59, 60, 65, 75, 77 to 86, 95 to 100, 102 to 108, 115 to119, 121 to 130, 137, or a combination of two or more thereof.Embodiments of first amplification oligomers include the amplificationoligomers comprising, consisting or consisting essentially of a targethybridizing sequence selected from the group consisting of SEQ IDNOS:50, 51, 59, 60, 65, 75, 77 to 86, 102 to 108, 121 to 130, or acombination of two or more thereof. Embodiments of second amplificationoligomers include the amplification oligomers comprising, consisting orconsisting essentially of a target hybridizing sequence selected fromthe group consisting of SEQ ID NOS:57, 95 to 100, 115 to 119, 137, or acombination of two or more thereof. In one embodiment, there is providedan amplification oligonucleotide that is from 18 to 29 nucleobases inlength wherein the 18 to 29 nucleobases are all selected from contiguousnucleobases within SEQ ID NO:77. In one embodiment, there is provided anamplification oligonucleotide that is from 18 to 27 nucleobases inlength wherein the 18 to 27 nucleobases are all selected from contiguousnucleobases within SEQ ID NO:95. In one embodiment, there is provided anamplification oligonucleotide that is from 18 to 35 nucleobases inlength wherein the 18 to 35 nucleobases are all selected from contiguousnucleobases within SEQ ID NO:96. In one embodiment, there is provided anamplification oligonucleotide that is from 18 to 27 nucleobases inlength wherein the 18 to 27 nucleobases are all selected from contiguousnucleobases within SEQ ID NO:115. In one embodiment, there is providedan amplification oligonucleotide that is from 18 to 27 nucleobases inlength wherein the 18 to 27 nucleobases are all selected from contiguousnucleobases within SEQ ID NO:137. In one embodiment, one or more of theamplification oligomers for amplifying an HRV target nucleic acidcomprises at least one of a 5-Me-dC, non-Watson Crick base, degeneratebase, or combination thereof.

Probes for the specific detection of HRV sequences include oligomersselected from the group consisting of SEQ ID Nos. 48, 49, 54, 87 to 94,109 to 114, 131 to 136, or a combination of two or more thereof. In oneembodiment, one or more of the detection probe oligomers for detectingan HRV target nucleic acid (including an HRV amplicon) comprises atleast one of a 5-Me-dC, non-Watson Crick base, degenerate base, orcombination thereof.

Assays for detection of Adeno, hMPV, and/or HRV nucleic acid may includean internal control (IC) nucleic acid that is amplified and detected byusing IC-specific primers and probe in the same reaction mixtures usedfor target nucleic acid amplification and detection Amplification anddetection of the IC-specific sequence demonstrates that assay reagentsand conditions were properly used even when no target-specific signal isdetected for a tested sample (i.e., negative samples). The IC may beused as an internal calibrator for the assay that provides aquantitative result. The IC may be a randomized sequence derived from anaturally occurring source that is not a target nucleic acid.

Sample Preparation

Preparation of samples for amplification and detection of target nucleicacid sequences may include methods of separating and/or concentratingorganisms contained in a sample from other sample components. Samplepreparation may include routine methods of disrupting samples or lysingsamples to release intracellular contents, including target nucleicacids or genetic sequences comprising target nucleic acid. Samplepreparation before amplification may include an optional step of targetcapture to specifically or non-specifically separate the target nucleicacids from other sample components. Nonspecific target capture methodsmay involve selective precipitation of nucleic acids from asubstantially aqueous mixture, adherence of nucleic acids to a supportthat is washed to remove other sample components, other methods ofphysically separating nucleic acids from a mixture that containsAdenovirus nucleic acid and other sample components.

Amplification of the Adenovirus Target Region

Amplifying a target nucleic acid target region using two or more primersmay be accomplished using a variety of known nucleic acid amplificationreactions. For example, amplification may be achieved using PCRamplification (U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159,Mullis et al.) to produce multiple DNA strands by using thermocyclingreactions that separate dsDNA and primers specific for portions of theseparated strands to make additional dsDNA molecules by using a DNApolymerase. Well known variations of the basic PCR method may also beused, e.g., PCR coupled with real-time detection—such as Taqman PCR.

Nucleic Acid Detection

Detection of the nucleic acids may be accomplished by a variety ofmethods. Detection methods may use nucleic acid probes comprising atarget hybridizing sequence that is complementary to a portion of theamplified product and detecting the presence of the probe:productcomplex, or by using a complex of probes that may amplify the detectablesignal associated with the amplified products (e.g., U.S. Pat. Nos.5,424,413; 5,451,503; 5,849,481; 5,639,604 and 5,283,174). Directly orindirectly labeled probes that specifically associate with the amplifiedproduct provide a detectable signal that indicates the presence of thetarget nucleic acid in the sample. For example, if the target nucleicacid is Adenovirus DNA, the amplified product will contain a sequence inor complementary to an Adenovirus target sequence. A probe is configuredto bind directly or indirectly to a portion of the amplification productto indicate the presence of Adenovirus in the tested sample.

In embodiments that detect the amplified product near or at the end ofthe amplification step, a linear detection probe may be used to providea signal to indicate hybridization of the probe to the amplifiedproduct. One example of such detection uses a luminescentally labeledprobe that hybridizes to target nucleic acid Luminescent label is thenhydrolyzed from non-hybridized probe. Detection is performed bychemiluminescence using a luminometer (see, e.g., International PatentApplication Pub. No. WO 89/002476). In other embodiments that usereal-time detection, the detection probe may be a hairpin probe such as,for example, a molecular beacon, molecular torch, or hybridizationswitch probe that is labeled with a reporter moiety that is detectedwhen the probe binds to amplified product. Such probes may comprisetarget-hybridizing sequences and non-target-hybridizing sequences.Various forms of such probes are described, e.g., in U.S. Pat. Nos.5,118,801; 5,312,728; 5,925,517; 6,150,097; 6,849,412; 6,835,542;6,534,274; and 6,361,945; and US Patent Application Pub. Nos.20060068417A1 and 20060194240A1).

Homogeneously detectable labels, such as fluorescent labels andelectronically detectable labels, are intended for use in the practiceof the present disclosure. Examples of detectable labels that arepreferred as members of an interactive pair of labels interact with eachother by FRET or non-FRET energy transfer mechanisms. Fluorescenceresonance energy transfer (FRET) involves the radiationless transmissionof energy quanta from the site of absorption to the site of itsutilization in the molecule, or system of molecules, by resonanceinteraction between chromophores, over distances considerably greaterthan interatomic distances, without conversion to thermal energy, andwithout the donor and acceptor coming into kinetic collision. The“donor” is the moiety that initially absorbs the energy, and the“acceptor” is the moiety to which the energy is subsequentlytransferred, hi addition to FRET, there are at least three other“non-FRET” energy transfer processes by which excitation energy can betransferred from a donor to an acceptor molecule.

When two labels are held sufficiently close that energy emitted by onelabel can be received or absorbed by the second label, whether by a FRETor non-FRET mechanism, the two labels are said to be in “energy transferrelationship” with each other, wherein fluorescent emission from afluorophore attached to one portion of the probe is quenched by aquencher moiety on another portion of the probe (e.g., “Taqman”detection probe chemistry). Label moieties for the Taqman probes includea fluorophore and a second moiety having fluorescence quenchingproperties (i.e., a “quencher”). In this embodiment, the characteristicsignal is likely fluorescence of a particular wavelength, butalternatively could be a visible light signal. When fluorescence isinvolved, changes in emission are preferably due to FRET, or toradiative energy transfer or non-FRET modes. When a detection probehaving a pair of interactive labels in the “quenched” state isstimulated by an appropriate frequency of light, a fluorescent signal isgenerated at a first level, which may be very low. When this same probeis in an “unquenched” state and is stimulated by an appropriatefrequency of light, the fluorophore and the quencher moieties aresufficiently separated from each other that energy transfer between themis substantially precluded. Under that condition, the quencher moiety isunable to quench the fluorescence from the fluorophore moiety. If thefluorophore is stimulated by light energy of an appropriate wavelength,a fluorescent signal of a second level, higher than the first level,will be generated. The difference between the two levels of fluorescenceis detectable and measurable. Examples of fluorophore/quencher labelpairs that may be used in connection with the disclosure, includefluorescein/tetramethylrhodamine, IAEDANS/fluororescein, EDANS/D ABCYL,coumarin/D ABCYL, fluorescein/fluorescein, BODIPY FL/BODIPY FL,fluorescein/D ABCYL, lucifer yellow/D ABCYL, BODIPY/D ABCYL, eosine/DABCYL, erythrosine/D ABCYL, tetramethylrhodamine/D ABCYL,CalOrange/BHQ1, CalRed/BHQ2, Texas Red/DABCYL, CY5/BH1, CY5/BH2,CY3/BH1, CY3/BH2, fluorescein/QSY7, FAM/BHQ1 and Quasar/BHQ2. Thosehaving an ordinary level of skill in the art will understand that whendonor and acceptor dyes are different, energy transfer can be detectedby the appearance of sensitized fluorescence of the acceptor or byquenching of donor fluorescence. When the donor and acceptor species arethe same, energy can be detected by the resulting fluorescencedepolarization. Non-fluorescent acceptors such as DABCYL and the QSY 7dyes advantageously eliminate the potential problem of backgroundfluorescence resulting from direct (i.e., non-sensitized) acceptorexcitation.

Synthetic techniques and methods of bonding labels to nucleic acids anddetecting labels are well known in the art (e.g., see Sambrook et al.,Molecular Cloning. A Laboratory Manual. 2nd ed. (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989), Chapter 10; Nelson etal., U.S. Pat. No. 5,658,737; Woodhead et al., U.S. Pat. No. 5,656,207;Hogan et al., U.S. Pat. No. 5,547,842; Arnold et al., U.S. Pat. No.5,283,174; Kourilsky et al., U.S. Pat. No. 4,581,333), and Becker etal., European Patent App. No. 0 747 706.

Kits

The oligomers for use in the methods described herein are suited forpreparation of kits. Such a kit may comprise containers, each with oneor more of the various oligomers optionally together with one or more ofthe reagents (eg. enzymes) required to perform the methods describedherein. The components of the kit may be supplied in concentrated form.A set of instructions for using the components of the kit will alsotypically be included. Where the kit comprises combinations of oligomersthen the individual oligomers may be provided in individual form, withappropriate instructions for mixing same, or combinations thereof thatare ready mixed.

In one aspect, there is provided a kit comprising the composition of thepresent disclosure and optionally a set of instructions for performingsame.

Correlation of Detection of a Target Sequence with Diagnosis

The detection of amplified target sequences characteristic ofAdenovirus, Metapneumovirus, and/or Rhinovirus in a biological samplefrom an individual is indicative, respectively, of infection byAdenovirus, Metapneumovirus, and/or Rhinovirus.

EXAMPLES Example 1: Analysis of Certain Adenovirus Amplification Primersand Probes Materials & Methods

In a first amplification reaction, the following was used: Fast StartMaster Buffer (Roche) at 1× to 2× concentration, 2 Units of Fast StartTaq DNA polymerase (Roche), 100 nM of a forward amplification primer(SEQ ID No. 5) and 100 nM of a reverse amplification primer (SEQ ID No.6 or SEQ ID No. 8) and 100 nM probe (SEQ ID No. 10).

The total reaction volume was 20 microlitres with 5 microlitres oftemplate nucleic acid extracted from Adenovirus added per reaction.Control reactions were performed by setting up a reaction as describedabove but not adding any template nucleic acids. The amplificationcycles used were as follows for both sets of amplification reactions:Hold for 600 seconds at 95 deg. C. with optics off; 95 deg. C. for 30seconds with optics off and 55 deg. C. for 60 seconds with optics on (5cycles); 95 deg. C. for 10 seconds with optics off and 55 deg. C. for 60seconds (40 cycles) with optics on.

Results

TABLE 1 Adenovirus Amplification and Detection with Primer and ProbeSets SEQ ID Nos. 5, 6 and 10 SEQ ID Nos. 5, 8 and 10 C_(T) RFU C_(T) RFUTarget/Sample 26.9 519 26.4 383

The results are presented as CTIRFU (cycle threshold/relativefluorescent unit) values and represent the average of 12 experimentsusing various Adenovirus serotypes. Amplification was not seen in any ofthe control reactions.

Conclusion

The primers and probes used appeared to be sensitive and specific forAdenovirus nucleic acid.

Example 2: Analysis of Further Certain Adenovirus Amplification Primersand Probes Materials & Methods

The following reagents were used: Fast Start Master Buffer (Roche) at 1×to 2× concentration, 2 Units of Fast Start Taq DNA polymerase (Roche),200 nM of a forward amplification primer (SEQ ID No. 11 or SEQ ID No.12) and 200 nM of a reverse amplification primer (SEQ ID No. 13 or SEQID No: 15) and 200 nM probe (SEQ ID No. 17 or SEQ ID No. 19).

The total reaction volume was 20 microlitres with 5 microlitres oftemplate nucleic acid extracted from Adenovirus added per reaction.Control reactions were performed by setting up a reaction as describedabove but not adding any template nucleic acids. The amplificationcycles used were as follows: Hold for 600 seconds at 95 deg. C. withoptics off; 95 deg. C. for 30 seconds with optics off and 55 deg. C. for60 seconds with optics on (5 cycles); 95 deg. C. for 10 seconds withoptics off and 55 deg. C. for 60 seconds (40 cycles) with optics on.

Results

TABLE 2 Adenovirus Amplification and Detection with Primer and ProbeSets SEQ ID Nos. 11, SEQ ID Nos. 11, 13 and 17 13 and 19 C_(T) RFU C_(T)RFU Target/Sample 8.7 29.8 32 460 SEQ ID Nos. 11, SEQ ID Nos. 11, 15 and17 15 and 19 C_(T) RFU C_(T) RFU Target/Sample 13.3 29.5 32.3 406.4 SEQID Nos. 12, SEQ ID Nos. 12, 15 and 19 15 and 17 C_(T) RFU C_(T) RFUTarget/Sample 29.6 620 12.6 32.8 SEQ ID Nos. 12, SEQ ID Nos. 12, 13 and19 13 and 17 C_(T) RFU C_(T) RFU Target/Sample 29.6 504 8.3 18.9

The results are presented as CTIRFU values and represent the average of8 experiments using various Adenovirus serotypes. Amplification was notseen in any of the control reactions.

Conclusion

Combinations of SEQ ID Nos. 11, 13 and 19, SEQ ID Nos. 11, 15 and 19,SEQ ID Nos. 12, 15 and 19 or SEQ ID Nos. 12, 13 and 19 were sensitiveand specific for Adenovirus nucleic acid. The combinations comprisingthe SEQ ID No. 12 forward primer appears to have better sensitivity thanthe combination comprising the SEQ ID No. 11 forward primer. Thecombination comprising SEQ ID Nos. 12, 15 and 19 appeared to be mostsensitive in these experiments.

Example 3: Adenovirus Serotype Analysis Using SEQ ID Nos. 12, 15 and 19Materials & Methods

The following reagents used: Fast Start Master Buffer (Roche) at 1×concentration, 2 Units of Fast Start Taq DNA polymerase (Roche), 400 nMof a forward amplification primer (SEQ ID No. 12) and 400 nM of areverse amplification primer (SEQ ID No. 15) was used together with 400nM probe (SEQ ID No. 19). The total reaction volume was 20 microlitreswith 5 microlitres of template nucleic acid extracted from Adenovirusadded per reaction. Control reactions were set-up, but no templatenucleic acid was added. The amplification cycles used were as follows:Hold for 600 seconds at 95 deg. C. with optics off; 95 deg. C. for 30seconds with optics off and 55 deg. C. for 60 seconds with optics on (5cycles); 95 deg. C. for 10 seconds with optics off and 55 deg. C. for 60seconds (40 cycles) with optics on.

Results

TABLE 3 Adenovirus Serotype Analysis Serotype C_(T) RFU  2-1 26 1203 4-1 31 666  6-1 29 926  7-1 32 605  9-1 26 1137 10-1 28 1252 11-1 28630 12-1 26 1119 13-1 26 1100 14-1 30 682 15-1 26 1078 16-1 29 723 17-123 1100 18-1 34 387 19-1 27 1146 20-1 23 996 21-1 31 568 22-1 25 104423-1 23 1109 24-1 25 1221 25-1 32 836 26-1 24 1107 27-1 25 1070 28-1 26989 29-1 27 1116 30-1 22 1166 31-1 21 1127 33-1 28 941 34-1 28 654 35-129 542 36-1 24 997 37-1 24 1125 38-1 26 1033 39-1 23 1143 40-1 27 111441-1 25 994 42-1 23 1125 43-1 22 1149 44-1 22 1141 45-1 27 1071 46-1 271047 47-1 22 1144 48-1 25 1174 49-1 26 1068 50-1 25 672 51-1 26 1099 1-1 29 956  3-1 32 540  5-1 29 791 7A-1  26 632  8-1 34 553 32-2 24 974

The Serotype column is set-up to reflect “serotype number-1×10^(x)TCID₅₀/mL.” C_(T) values have all been rounded down. The results arepresented as C_(T)/RFU values.

Conclusion

The combination of SEQ ID Nos. 12, 15 and 19 was able to detect allserotypes of Adenovirus that were tested.

Example 4: Analysis of Further Adenovirus Probe Combinations Togetherwith SEQ ID NOS: 12 and 15 Primers Materials & Methods

The following reagents used: Fast Start Master Buffer (Roche) at 1×concentration, 2 Units of Fast Start Taq DNA polymerase (Roche), 100 nMof a forward amplification primer (SEQ ID No. 12) and 100 nM of areverse amplification primer (SEQ ID No. 15) was used together witheither: 150 nM probe (SEQ ID No. 21) and 50 nM probe (SEQ ID No. 24);100 nM probe (SEQ ID No. 21) and 100 nM probe (SEQ ID No. 24); 50 nMprobe (SEQ ID No. 21) and 150 nM probe (SEQ ID No. 24). The totalreaction volume was 20 microlitres with 5 microlitres of templatenucleic acid extracted from Adenovirus added per reaction. Controlreactions were set-up without the addition of template nucleic acid. Theamplification cycles used were as follows: Hold for 600 seconds at 95deg. C. with optics off; 95 deg. C. for 30 seconds with optics off and55 deg. C. for 60 seconds with optics on (5 cycles); 95 deg. C. for 10seconds with optics off and 55 deg. C. for 60 seconds (40 cycles) withoptics on.

Results

TABLE 4 Amplification and detection using different concentrations ofprobe combinations 150 nM SEQ ID No. 100 nM SEQ ID No. 21 and 50 nM 21and 100 nM SEQ ID No. 24; SEQ ID No. 24; C_(T) RFU C_(T) RFU Target 34.8291 26.9 318 50 nM SEQ ID No. 21 and 150 nM SEQ ID No. 24 C_(T) RFUTarget 26.8 339

The results are presented as CTIRFU values and represent the average of6 experiments using various Adenovirus serotypes.

Conclusion

SEQ ID No. 21 and ID No. 24 probes in combination with SEQ ID No. 12 and15 were able to sensitively and specifically detect Adenovirus at thevarious concentrations tested.

Example 5: Analysis of Further Probe and Primer Combinations for theDetection of Adenovirus Materials & Methods

The following reagents used:

Fast Start Master Buffer (Roche) at 1× concentration, 2 Units of FastStart Taq DNA polymerase (Roche) and either: (i) 50 mM of a forwardamplification primer (SEQ ID No. 25), 50 mM of a forward amplificationprimer (SEQ ID No. 26), 50 mM of a reverse amplification primer (SEQ IDNo. 27), 50 mM of a reverse amplification primer (SEQ ID No. 28) and 100nM of probes (SEQ ID No. 21 and SEQ ID No. 23); (ii) 50 mM of a forwardamplification primer (SEQ ID No. 26), 50 mM of a reverse amplificationprimer (SEQ ID No. 27), 50 mM of a reverse amplification primer (SEQ IDNo. 28) and 100 nM of probes (SEQ ID No. 21 and SEQ ID No. 23); (iii) 50mM of a forward amplification primer (SEQ ID No. 25), 50 mM of a reverseamplification primer (SEQ ID No. 27), 50 mM of a reverse amplificationprimer (SEQ ID No. 28) and 100 nM of probes (SEQ ID No. 21 and SEQ IDNo. 23); (iv) 50 mM of a forward amplification primer (SEQ ID No. 25),50 mM of a forward amplification primer (SEQ ID No. 26), 50 mM of areverse amplification primer (SEQ ID No. 28) and 100 nM of probes (SEQID No. 21 and SEQ ID No. 23); (v) 50 mM of a forward amplificationprimer (SEQ ID No. 25), 50 mM of a forward amplification primer (SEQ IDNo. 26), 50 mM of a reverse amplification primer (SEQ ID No. 28) and 100nM of probes (SEQ ID No. 21 and SEQ ID No. 23); (vi) 50 mM of a forwardamplification primer (SEQ ID No. 25), 50 mM of a forward amplificationprimer (SEQ ID No. 26), 50 mM of a reverse amplification primer (SEQ IDNo. 27), 50 mM of a reverse amplification primer (SEQ ID No. 28) and 100nM of probe (SEQ ID No. 23); or (vii) 50 mM of a forward amplificationprimer (SEQ ID No. 25), 50 mM of a forward amplification primer (SEQ IDNo. 26), 50 mM of a reverse amplification primer (SEQ ID No. 27), 50 mMof a reverse amplification primer (SEQ ID No. 28) and 100 nM of probes(SEQ ID No. 21).

The total reaction volume was 20 microlitres with 5 microlitres oftemplate nucleic acid extracted from Adenovirus added per reaction. Twodifferent concentrations were tested.

The amplification cycles used were as follows: Hold for 600 seconds at95 deg. C. with optics off; 95 deg. C. for 30 seconds with optics offand 55 deg. C. for 60 seconds with optics on (5 cycles); 95 deg. C. for10 seconds with optics off and 55 deg. C. for 60 seconds (40 cycles)with optics on.

Results

Tables 5a-5d. Amplification and detection using different concentrationsand combinations of primers and probes.

TABLE 5a SEQ ID Nos. 25, 26, SEQ ID Nos. 26, 27, 28, 21 and 23 27, 28,21 and 23 C_(T) RFU C_(T) RFU Target(10¹) 38.5 240 39.2 143 Target(10³)29.8 373 30.8 227

TABLE 5b SEQ ID Nos. 25, SEQ ID Nos. 25, 27, 28, 21 and 23 26, 28, 21and 23 C_(T) RFU C_(T) RFU Target(10¹) 37.8 212 41.5 99 Target(10³) 30.2275 32 258

TABLE 5c SEQ ID Nos. 25, SEQ ID Nos. 25, 26, 27, 21 and 23 26, 27, 28and 23 C_(T) RFU C_(T) RFU Target(10¹) 41.8 96 37.7 254 Target(10³) 31.8320 30 360

TABLE 5d SEQ ID Nos. 25, 26, 27, 28 and 21 C_(T) RFU Target (10¹) 7.1 21Target (10³) 0 3

The results are presented as RFU values and represent the average of 6experiments for each concentration.

Conclusion

Leaving out one of the primers or probes from the assay made littledifference for the most part. However, omitting probe SEQ ID No. 23resulted in lower detection in this particular experiment.

Example 6: Analysis of Primer and Probe Combinations for DetectingAdenovirus 18 Materials & Methods

The following reagents used: Fast Start Master Buffer (Roche) at 1×concentration, 3 Units of Fast Start Taq DNA polymerase (Roche), 150 nMforward amplification primers (SEQ ID No. 25 and SEQ ID No. 26) and 150nM reverse amplification primers (SEQ ID No. 27 and SEQ ID No. 28) wereused together with 300 nM probe (SEQ ID No. 29). The total reactionvolume was 20 microlitres with 5 microlitres of template nucleic acidextracted from Adenovirus 18 added per reaction. The amplificationcycles used were as follows: Hold for 600 seconds at 95 deg. C. withoptics off; 95 deg. C. for 30 seconds with optics off and 55 deg. C. for60 seconds with optics on (5 cycles); 95 deg. C. for 10 seconds withoptics off and 55 deg. C. for 60 seconds (40 cycles) with optics on.

Results

TABLE 6 Amplification and detection of Adenovirus 18. Serotype C_(T) RFU18-6 17 1240 18-5 20 975 18-4 24 1242 18-3 30 1023 18-2 33 942 18-1 35747 18-0 31 1215

The Serotype column is set-up to reflect “serotype number-1×10^(x)TCID₅₀/mL.” C_(T) values have all been rounded down. The results arepresented as C_(T)/RFU values.

Conclusion

This combination of primers and probes successfully detects Adenovirus18.

Example 7: Analysis of Further Primer and Probe Combinations forDetecting Adenovirus Materials & Methods

The following reagents used: Fast Start Master Buffer (Roche) at 1×concentration, 3 Units of Fast Start Taq DNA polymerase (Roche), 150 nMforward amplification primers (SEQ ID No. 31 and SEQ ID No. 26) and 150nM reverse amplification primers (SEQ ID No. 27 and SEQ ID No. 28) wereused together with 150 nM probe (SEQ ID No. 21 and SEQ ID No. 23). Thetotal reaction volume was 20 microlitres with 5 microlitres of templatenucleic acid extracted from various Adenovirus serotypes added perreaction. The amplification cycles used were as follows: Hold for 600seconds at 95 deg. C. with optics off; 95 deg. C. for 30 seconds withoptics off and 55 deg. C. for 60 seconds with optics on (5 cycles); 95deg. C. for 10 seconds with optics off and 55 deg. C. for 60 seconds (40cycles) with optics on.

Results

TABLE 7 Primer and probe combinations for detecting various Adenovirusserotypes. FAM Cy5 Serotype C_(T) RFU C_(T) RFU 1 38.7 40 35.9 172 337.4 59 35.5 208 4 0 22 37.2 185 7 32.2 711 32 209 11 24.2 843 32.2 19014 28.8 737 31.9 196 16 23.8 879 32.1 212 21 31.9 671 31.4 219 25 32.8399 31.7 217 34 29.3 645 31.7 205 35 29.6 771 30.7 220 50 24.6 786 30.9210

The results are presented as CT/RFU values. The Fam-channel showsdetection results for the template nucleic acids. The Cy5-channel showsdetection results for an internal control nucleic acid.

Conclusion

With the exception of serotype 4, this combination of primers and probessuccessfully detected all of the serotypes tested.

Example 8: Analysis of Further Primer and Probe Combinations forDetecting Adenovirus Materials & Methods

The following reagents used: Fast Start Master Buffer (Roche) at 1×concentration, 3 Units of Fast Start Taq DNA polymerase (Roche) andeither: (i) 150 nM forward amplification primers (SEQ ID Nos. 33 and 34)and 150 nM reverse amplification primers (SEQ ID No. 27 and SEQ ID No.28) were used together with 150 nM probe (SEQ ID No. 21 and SEQ ID No.23); (ii) 150 nM forward amplification primers (SEQ ID Nos. 33 and 35)and 150 nM reverse amplification primers (SEQ ID No. 27 and SEQ ID No.28) were used together with 150 nM probe (SEQ ID No. 21 and SEQ ID No.23); or (iii) 150 nM forward amplification primers (SEQ ID Nos. 34 and35) and 150 nM reverse amplification primers (SEQ ID No. 27 and SEQ IDNo. 28) were used together with 150 nM probe (SEQ ID No. 21 and SEQ IDNo. 23). The total reaction volume was 20 microlitres with 5 microlitresof template nucleic acid extracted from various Adenovirus serotypesadded per reaction. The amplification cycles used were as follows: Holdfor 600 seconds at 95 deg. C. with optics off; 95 deg. C. for 30 secondswith optics off and 55 deg. C. for 60 seconds with optics on (5 cycles);95 deg. C. for 10 seconds with optics off and 55 deg. C. for 60 seconds(40 cycles) with optics on.

Results

Tables 8a-8c. Amplification and detection of various Adenovirusserotypes using combinations of primers and probes.

TABLE 8a SEQ ID Nos. 33, 34, 27, 28, 21 and 23 FAM Cy5 Serotype C_(T)RFU C_(T) RFU 1 35.6 318 35.9 243 3 37.3 87 35.8 210 4 36.9 150 35.6 23219 38.1 95 35.2 217 31 35.7 247 34.6 257 41 36.7 244 35.9 285 14 29.7868 31.5 250

TABLE 8b SEQ ID Nos. 33, 35, 27, 28, 21 and 23 FAM Cy5 Serotype C_(T)RFU C_(T) RFU 1 0 12 35.7 248 3 36.4 159 35.1 231 4 36.8 171 35.6 249 1936.7 151 35.3 181 31 35.8 170 34.6 197 41 39.3 50 36 243 14 29.2 106231.5 256

TABLE 8c SEQ ID Nos. 34, 35, 27, 28, 21 and 23 FAM Cy5 Serotype C_(T)RFU C_(T) RFU 1 37.5 198 35.5 199 3 0 5 35.5 213 4 0 15 36.1 158 19 0 1235.1 224 31 35.6 369 34.6 240 41 36.6 284 35.7 263 14 33.1 942 32.1 203

The results are presented as C_(T) and RFU values. The Fam-channel showsdetection results for the template nucleic acids. The Cy5-channel showsdetection results for an internal control nucleic acid.

Conclusion

Table 8a of primers and probes successfully detected all of theserotypes tested. Tables 8b and 8c detected most serotypes tested.

Example 9: Analysis of Further Primer and Probe Combinations forDetecting Adenovirus Materials & Methods

The following reagents used: Fast Start Master Buffer (Roche) at 1×concentration, 3 Units of Fast Start Taq DNA polymerase (Roche) and 150nM forward amplification primers (SEQ ID Nos. 25 and 26) and 150 nMreverse amplification primers (SEQ ID No. 27 and SEQ ID No. 28) wereused together with 150 nM probe (SEQ ID No. 36 and SEQ ID No. 37). Thetotal reaction volume was 20 microlitres with 5 microlitres of templatenucleic acid extracted from the Adenovirus 19 serotype positive controlplasmid, which added per reaction at six different concentrations. Theamplification cycles used were as follows: Hold for 600 seconds at 95deg. C. with optics off; 95 deg. C. for 30 seconds with optics off and55 deg. C. for 60 seconds with optics on (5 cycles); 95 deg. C. for 10seconds with optics off and 55 deg. C. for 60 seconds (40 cycles) withoptics on.

Results

TABLE 9 Amplification and detection of a serial dilution of targetnucleic acid FAM Concentration C_(T) RFU 10⁴ 28.1 1127 10⁴ 28.2 1040 10⁴28 1196 10³ 31.9 938 10³ 32.1 922 10³ 32.3 969 10² 35.3 865 10² 35.4 80010² 35.2 800 10¹ 37.8 571 10¹ 33.6 59 10¹ 38.6 419 10⁰ 0 10 10⁰ 0 0 10⁰0 0 10⁻¹ 0 0 10⁻¹ 0 0 10⁻¹ 0 0The results are presented as CTIRFU values.

Conclusion

These primers and probes successfully detected the control testedAdenovirus 19 serotype.

Example 10: Further Analysis of the Primer and Probe Combination fromExample 9 Materials & Methods

The following reagents used: Fast Start Master Buffer (Roche) at 1×concentration, 3 Units of Fast Start Taq DNA polymerase (Roche) and 150nM forward amplification primers (SEQ ID Nos. 25 and 26) and 150 nMreverse amplification primers (SEQ ID No. 27 and SEQ ID No. 28) wereused together with 150 nM probe (SEQ ID No. 36 and SEQ ID No. 37). Thetotal reaction volume was 20 microlitres with 5 microlitres of templatenucleic acid extracted from various Adenovirus serotypes and tested at aconcentration of 3×10⁰. The amplification cycles used were as follows:Hold for 600 seconds at 95 deg. C. with optics off; 95 deg. C. for 30seconds with optics off and 55 deg. C. for 60 seconds with optics on (5cycles); 95 deg. C. for 10 seconds with optics off and 55 deg. C. for 60seconds (40 cycles) with optics on.

Results

TABLE 10 Amplification and detection of Adenovirus target nucleic acidsSerotype C_(T) RFU 2 31.6 1465 5 33.9 903 6 0 0 7 39 366 8 37.4 625 9 321223 10 36.3 837 11 26.6 950 12 31.8 1176 13 29.7 1487 14 34 671 15 33.51018 16 33.4 729 17 30.2 1622 18 40 175 20 27.2 1217 21 34.1 733 22 31.21150 23 30.1 1471 24 33.2 1110 25 37.3 661 26 29.1 1814 27 29.5 1637 2834.2 1032 29 32.9 1159 30 28.4 1496 32 26.6 2079 33 33.9 1017 34 34.2690 35 33.1 702 36 29.1 1312 37 30.2 1393 38 31.8 1202 39 30 1650 4032.6 1290 42 29.1 1261 43 28.8 1832 44 23.3 1218 45 32.1 1289 46 32.51183 47 26.4 1209 48 29.4 1565 49 31.5 1264 50 32 846 51 31.4 1125 Theresults are presented as C_(T) and RFU values.

Discussion

All of the serotypes tested were detected using this primer and probeconcentration with the exception of serotype 6. This serotype wassuccessfully detected at 3×10¹ TCID₅₀/mL and above.

Example 11. Multiplex Amplification and Detection of Adenovirus andHuman Metapneumovirus and Rhinovirus Materials & Methods

Analytical Sensitivity and Reactivity: A PCR formulation containingprimers and probes for the amplification and detection of Adenovirus,human Metapneumovirus, and Rhinovirus was prepared to include (perreaction): 15 uL of Supermix (11.1 Units Taq); 1.2 uL of MMLV reversetranscriptase (RT) (24 Units); 2.0 uL of Primer Probe Mix; 0.084 uL of0.5M EDTA; and 1.716 uL of Water. (AMR formulation.) Target nucleicacids were extracted from a number of stock organisms¹ and diluted to0.1 TCID₅₀/mL, 1 TCID₅₀/mL and 10 TCID₅₀/mL for each organism. 10 uL oftarget nucleic acid elute from each dilution was individually combinedwith a reaction volume of the PCR formulation for a 30 uL total reactionvolume. Primers and probes used in this experiment are shown in SEQ IDNOs:48-49, & 51-74, which include internal controls. Probes used in thisexample were dual labelled probes comprising quenchers and fluorophores.

A multiplex, real-time PCR reaction was set up using the Pantherinstrument (Hologic, Inc., San Diego, Calif.) for sample preparation andthe benchtop PCR thermocycler for real-time amplification and detection.Detection reactions used Taqman™ (Roche Molecular Systems, Inc.,Pleasanton, Calif.) chemistry. Adenovirus target nucleic acids weredetected in a ROX channel, hMPV nucleic acids were detected in a HEXchannel, HRV nucleic acids were detected in a FAM channel, and theinternal control was detected in a RED677 channel of the thermocycler.The assay was performed using 10 replicates of each viral target at theknown concentrations Each individual sample preparation was evaluated asone real time RT-PCR replicate on a benchtop PCR thermocycler. Positiveor negative determinations were made using background subtracted curves.Table 11 thru Table 13 demonstrate 100% hit rate at viral concentrationsat or below 10 TCID₅₀/mL.

Subsequent to these studies, two additional bases were added to the 5′end of primer SEQ ID NO:75 to generate primer SEQ ID NO:50. A Rhinovirussensitivity assay was performed as above with SEQ ID NO:50 in place ofSEQ ID NO:75 and the results are presented in Table 14.

TABLE 11 Rhinovirus detection using AdV/hMPV/RV combined oligonucleotideformulation Percent Rhino- Concentration Hit Rate Avg StDev StDev virus(TCID50/ml) (n = 10) Ct Ct RFU RFU Rhino- 10{circumflex over ( )}0 100%34.2 0.4 14828 4048 virus A-18 Rhino- 10{circumflex over ( )}0 100% 35.10.4 4641 662 virus B-26 ¹Target nucleic acids were isolated frompreviously characterized stock organisms obtained from TriCore ReferenceLaboratories (Albuquerque, NM); ZeptoMetrix Corporation (Buffalo, NY);and ATCC (Manassas, VA).

TABLE 12 hMPV detection using AdV/hMPV/RV combined oligonucleotideformulation Percent Concentration Hit Rate Avg StDev StDev hMPV(TCID50/ml) (n = 10) Ct Ct RFU RFU hMPV 10{circumflex over ( )}1 100%37.7 0.6 3019 630 A1-16 hMPV 10{circumflex over ( )}1 100% 34.8 0.3 6186583 A2-20 hMPV 10{circumflex over ( )}1 100% 35.8 1.0 5818 1357 B1-3hMPV 10{circumflex over ( )}1 100% 34.1 0.4 8043 1206 B2-8

TABLE 13 Adenovirus detection using AdV/hMPV/RV combined oligonucleotideformulation Percent Concentration Hit Rate Avg StDev StDev Adenovirus(TCID50/ml) (n = 10) Ct Ct RFU RFU AdV 1 10{circumflex over ( )}−1 100%38.1 0.8 2919 921 (Species C) AdV 3 10{circumflex over ( )}0  100% 36.10.3 5809 1157 (Species B) AdV 4 10{circumflex over ( )}−3 100% 37.9 1.92738 965 (Species E) AdV 9 10{circumflex over ( )}−1 100% 37.6 0.7 2786831 (Species D) AdV 12 10{circumflex over ( )}−1 100% 36.4 0.8 2186 549(Species A) AdV 40 10{circumflex over ( )}0  100% 38.5 1.4 2320 1184(Species F)

TABLE 14 Rhinovirus B detection using AdV/hMPV/RV combinedoligonucleotide formulation containing SEQ ID NO: 74 PercentConcentration Hit Rate Avg StDev StDev Rhinovirus (TCID50/ml) (n = 10)Ct Ct RFU RFU Rhinovirus 10{circumflex over ( )}0 100% 36.4 0.3 75091459 B-26

Conclusion:

A multiplexed combination of the amplification and detectionoligonucleotides presented in this review is capable of detecting viralconcentrations at or below 10 TCID₅₀/mL.

Example 12. Clinical Specimen Detection and Clinical Specificity Using aMultiplexed Amplification and Detection Assay Materials & Methods

PCR formulations (AMR formulation) containing all of the primers andprobes listed above in Example 11 (using SEQ ID NO:74) were used to testclinical specimens that had been identified to be Rhinovirus positive,hMPV positive, and/or Adenovirus positive or negative for all threeviruses by a commercial assay. The commercial assays include the BioFireFilmArray RVP Respiratory Panel (BioFire Diagnostics, Salt Lake City,Utah), the GenMark eSensor Respiratory Virus Panel (RVP) (GenMarkDiagnostics, Inc., Carlsbad, Calif.), and the Luminex xTAG RespiratoryVirus Panel (Luminex Corporation, Austin, Tex.). All samples wereextracted using the Panther instrument and PCR cycling was performed ona benchtop PCR thermocycler instrument as described above. The AMR assaydetected HRV, hMPV, and Adenovirus in these previously characterizedclinical specimens with a concordance of 94.8% (164/173) for HRV, 97.2%(279/287) for hMPV, and 93.2% (466/500) for Adenovirus. The AMR assayidentified 86 of 88 clinical specimen as negative, providing a 97.7%concordance with the reference assay (Luminex xTAG Respiratory VirusPanel). In addition the internal control was valid for all clinicallynegative specimens. The two ‘false positive’ results received using theAMR formulation assay were determined to be true positives using theGenMark eSensor RVP assay and the Prodesse ProAdeno+ Assay (Hologic,Inc., San Diego, Calif.). Thus, removing the clinical specimen falselyidentified as negative by the Luminex assay indicated a 100% concordanceby the AMR assay (86/86).

Conclusion:

A multiplexed combination of the amplification and detectionoligonucleotides presented in this example is capable of detecting viraltargets in clinical specimens and demonstrates good concordance withcompetitor assays.

Specificity Materials & Methods

A PCR formulation (AMR formulation) containing all of the primers andprobes listed in Example 11 (using SEQ ID NO:74) were evaluated forcross reactivity with other organisms. These organisms are thosecommonly found in specimen type (nasopharyngeal and lower respiratoryspecimens) that are tested in the clinic for the presence or absence ofone or more of Adenovirus, hMPV and HRV. Organisms were either pooledand tested or tested individually (see, AMR panels 1 thru 26 in Table15). Three replicates from each panel were individually processed on thePanther instrument and PCR cycling was performed on a benchtop PCRthermocycler instrument as described above. Table 15 demonstrates thatonly viruses targeted by the AMR formulation (AMR 24-26) were detected.Cross-reactivity with organisms not targeted by the assay (AMR 1-23) wasnot observed.

TABLE 15 Organisms and concentrations of the AMR formulation specificitypanel² Cross- Test Reactivity Panel Organism Concentration Results AMR 1Acinetobacter 1 × 10{circumflex over ( )}7.6 Not observed baumannii307-0294 CFU/ml Cornavirus 229E 1 × 10{circumflex over ( )}4.0 Notobserved TCID50/ml Bordetella parapertussis 1 × 10{circumflex over( )}7.2 Not observed CFU/ml Burkholderia cepacia 1 × 10{circumflex over( )}8.0 Not observed Z066 CFU/ml Candida albicans Z006 1 × 10{circumflexover ( )}6.5 Not observed CFU/ml Chlamydia pneumoniae 1 × 10{circumflexover ( )}5.8 Not observed CFU/ml AMR 2 Bordetella pertussis 1 ×10{circumflex over ( )}7.6 Not observed CFU/ml Candida glabrata Z007 1 ×10{circumflex over ( )}6.9 Not observed CFU/ml Chlamydia trachomatis 1 ×10{circumflex over ( )}5.8 Not observed CFU/ml Corynebacterium 1 ×10{circumflex over ( )}7.9 Not observed diphtheriae Z116 CFU/mlCornavirus NL63 1 × 10{circumflex over ( )}3.5 Not observed TCID50/mlCytomegalovirus AD- 1 × 10{circumflex over ( )}3.9 Not observed 169,MRC-5 TCID50/ml AMR 3 E. coli ETEC; ST+, 1 × 10{circumflex over ( )}7.8Not observed LT+ CFU/ml HPIV-1 1 × 10{circumflex over ( )}3.7 Notobserved TCID50/ml HSV-1 Macinytre Strain 1 × 10{circumflex over ( )}5.8Not observed TCID50/ml Cornavirus OC43 1 × 10{circumflex over ( )}5.3Not observed TCID50/ml IA/California/07/2009 1 × 10{circumflex over( )}3.5 Not observed 2009 H1N1 TCID50/ml Lactobacillus 1 × 10{circumflexover ( )}6.7 Not observed acidophilus Z048 CFU/ml AMR 4 HPIV-2 1 ×10{circumflex over ( )}5.6 Not observed TCID50/ml Neisseria elongata 1 ×10{circumflex over ( )}8.1 Not observed Z071 CFU/ml RSV A 1 ×10{circumflex over ( )}6.1 Not observed TCID50/ml IA/Massachusetts/15/131 × 10{circumflex over ( )}3.4 Not observed 2009 H1N1 TCID50/mlLegionella pneumophila 1 × 10{circumflex over ( )}8.5 Not observedPhiladelphia CFU/ml Mycobacterium 1 × 10{circumflex over ( )}5.6 Notobserved inracellular lysate CFU/ml AMR 5 HPIV-3 1 × 10{circumflex over( )}6.1 Not observed TCID50/ml HSV-2 Type 2G Strain 1 × 10{circumflexover ( )}4.8 Not observed TCID50/ml IA/Victoria/361/2011 1 ×10{circumflex over ( )}3.5 Not observed TCID50/ml Staphylococcus aureus1 × 10{circumflex over ( )}7.9 Not observed MSSA CFU/ml Epstein-BarrVirus 1 × 10{circumflex over ( )}6.8 Not observed B95-8 copies/mlHaemophilus Influenzae 1 × 10{circumflex over ( )}7.2 Not observed typeb; Eagan CFU/ml AMR 6 HPIV-4a 1 × 10{circumflex over ( )}4.0 Notobserved TCID50/ml IA/Switzerland/9715293/ 1 × 10{circumflex over( )}3.4 Not observed 2013 H3N2 TCID50/ml Mycoplasma pneumoniae 1 ×10{circumflex over ( )}6.1 Not observed M129 CFU/ml Streptococcuspyogenes 1 × 10{circumflex over ( )}7.7 Not observed Z018 CFU/mlStaphylococcus 1 × 10{circumflex over ( )}7.3 Not observed haemolyticusZ067 CFU/ml Lactobacillus plantarum 1 × 10{circumflex over ( )}7.2 Notobserved 17-5 CFU/ml AMR 7 Legionella micdadei 1 × 10{circumflex over( )}7.7 Not observed Tatlock CFU/ml IB/Brisbane/33/08  1 × 10{circumflexover ( )}3.45 Not observed TCID50/ml Staphylococcus 1 × 10{circumflexover ( )}7.8 Not observed epidermidis MRSE TCID50/ml Streptococcus 1 ×10{circumflex over ( )}7.2 Not observed agalactiae CFU/ml Klebsiellapneumonia 1 × 10{circumflex over ( )}8.1 Not observed Z026 CFU/ml AMR 8Measles Virus 1 × 10{circumflex over ( )}3.7 Not observed TCID50/mlMoraxella catarrhalis 1 × 10{circumflex over ( )}5.3 Not observed Ne 11CFU/ml IB/Massachusetts/2/2012 1 × 10{circumflex over ( )}3.5 Notobserved TCID50/ml Streptococcus 1 × 10{circumflex over ( )}6.2 Notobserved pneumoniae CFU/ml Mumps Virus 1 1 × 10{circumflex over ( )}4.3Not observed TCID50/ml AMR 9 Mycobacterium 1 × 10{circumflex over( )}5.4 Not observed tuberculosis lysate TCID50/ml Neisseria 1 ×10{circumflex over ( )}7.2 Not observed meningitidis A CFU/ml Mycoplasmahominis 1 × 10{circumflex over ( )}3.3 Not observed CFU/ml RSV B 1 ×10{circumflex over ( )}6.4 Not observed TCID50/ml Streptococcus 1 ×10{circumflex over ( )}6.7 Not observed salivarius CFU/ml AMR 10Pseudomonas 1 × 10{circumflex over ( )}8.3 Not observed aeruginosa Z139CFU/ml Serratia 1 × 10{circumflex over ( )}7.9 Not observed marcescensZ053 CFU/ml Streptococcus 1 × 10{circumflex over ( )}7.3 Not observedsanguinis Z089 CFU/ml Ureaplasma 1 × 10{circumflex over ( )}8.0 Notobserved urealyticum CFU/ml Varicella Zoster 1 × 10{circumflex over( )}3.5 Not observed Virus Ellen TCID50/ml AMR 11 Coxsackie B3 1 ×10{circumflex over ( )}6.6 Not observed TCID50/ml AMR 12 Coxsackie B4 1× 10{circumflex over ( )}3.8 Not observed TCID50/ml AMR 13 CoxsackieB5/10/2006 1 × 10{circumflex over ( )}5.8 Not observed TCID50/ml AMR 14Coxsackievirus A10 1 × 10{circumflex over ( )}3.7 Not observed TCID50/mlAMR 15 Coxsackievirus A21 1 × 10{circumflex over ( )}3.9 Not observedTCID50/ml AMR 16 Echovirus 6 1 × 10{circumflex over ( )}6.3 Not observedTCID50/ml AMR 17 Enterovirus 11 1 × 10{circumflex over ( )}6.8 Notobserved TCID50/ml AMR 18 Enterovirus 2 1 × 10{circumflex over ( )}6.8Not observed TCID50/ml AMR 19 Enterovirus 3 1 × 10{circumflex over( )}4.8 Not observed TCID50/ml AMR 20 Enterovirus 68 1 × 10{circumflexover ( )}2.8 Not observed TCID50/ml AMR 21 Enterovirus 70 1 ×10{circumflex over ( )}3.8 Not observed TCID50/ml AMR 22 Enterovirus 711 × 10{circumflex over ( )}6.2 Not observed TCID50/ml AMR 23 Poliovirus1 1 × 10{circumflex over ( )}6.3 Not observed TCID50/ml AMR 24Adenovirus 1 1 × 10{circumflex over ( )}6.3 17.3 AdV Ct TCID50/ml hMPV-3Subtype B1 1 × 10{circumflex over ( )}6.1 22.9 hMPV Ct TCID50/mlRhinovirus 1A 1 × 10{circumflex over ( )}4.4 22.5 RV Ct TCID50/ml AMR 25Adenovirus 4 1 × 10{circumflex over ( )}3.7 21.4 AdV Ct TCID50/ml hMPV-9Subtype A1 1 × 10{circumflex over ( )}7.5 19.1 hMPV Ct TCID50/mlRhinovirus 26B 1 × 10{circumflex over ( )}3.7 24.0 RV Ct TCID50/ml AMR26 Adenovirus 7a 1 × 10{circumflex over ( )}5.6 16.9 AdV Ct TCID50/ml²Target nucleic acids were isolated from previously characterized stockorganisms obtained from TriCore Reference Laboratories (Albuquerque,NM); ZeptoMetrix Corporation (Buffalo, NY); and ATCC (Manassas, VA). Ct= cycle time.

Conclusion:

A multiplexed combination of the amplification and detectionoligonucleotides presented in this review exhibits specificity for thetargets of the AdV/hMPV/RV assay.

As described above in Example 11, SEQ ID NO:50 was subsequentlysubstituted by SEQ ID NO:75 in the multiplex reagent. This modified PCRreaction formulation was tested in an amplification and detectionreaction using clinical samples and challenge organisms. The modifiedAMR formulation provided results similar to those shown in this Example12 (data not shown).

Exemplary Nucleic Acid Sequences.

The instant Table 16 provides exemplary sequences that are useful withthe present disclosure. This table does not limit the scope of thedisclosure. Sequences are presented according to World IntellectualProperty Organization (WIPO) Handbook on Industrial Property Informationand Documentation, Standard ST.25 (1998), including Tables 1 through 6of Appendix 2.

TABLE 16 Exemplary nucleic acid sequences SEQ ID No Sequence 5′ > 3′   1CAGGACGCCTCGGRGTAYCTSAG   2 GGAGCCACVGTGGGRTT   3 AAYCCCACBGTGGCTCC   4CCGGGTCTGGTGCAGTTTGCCCGC   5 CACATCGCCGGACAGGA   6 CATACTGAAGTAGGTGTCTGT  7 ACAGACACCTACTTCAGTATG   8 CGGTGGTCACATCGTGG   9 CCACGATGTGACCACCG 10 AGTACCTCAGTCCGGGTCTGGTG  11 ATGGCTACCCCTTCGATG  12 ACCCCMTCGATGATGCC 13 GCGGGCGAATTGCACCA  14 TGGTGCAATTCGCCCGC  15 GCGGGCAAAYTGCACCA  16TGGTGCARTTTGCCCGC  17 GACTCAGGTACTCCGAAGCATCCT  18AGGATGCTTCGGAGTACCTGAGTC  19 CTCAGGTACTCCGAGGCGTCCT  20AGGACGCCTCGGAGTACCTGAG  21 CTCAGGTACTCCGAAGCATCCT  22AGGATGCTTCGGAGTACCTGAG  23 CAGGTACTCCGAGGCGTCCT  24 AGGACGCCTCGGAGTACCTG 25 ACCCCATCGATGATGCC  26 ACCCCCTCGATGATGCC  27 GCGGGCAAACTGCACCA  28GCGGGCAAATTGCACCA  29 CTCAGGTATTCCGAGGCATCCT  30 AGGATGCCTCGGAATACCTGAG 31 ACCCCATCGATGCTGCC  32 ACCCCATCGATGATGCC  33 TGGGCGTACATGCACATC  34GTGGTCTTACATGCACATC  35 GTGGGCATACATGCACATC  36 AGGATGCTTCGGAGTACCTGAG 37 AGGACGCCTCGGAGTACCTG  38 ARTGGKCDTACATGCACATC  39CAGGACGCCTCGGAGTACCT  40 AGGATGCTTCGGAGTACCTGAG  41 CACGATGTGACCACAGA 42 CAYGATGTGACCACAGA  43 CACGAYGTGACCACAGA  44 CACGATGTGACCACSGA  45CACGATGTGACCACVGA  46 CAYGAYGTGACCACVGA  47Human adenovirus 9 gene for hexon, complete cds AB330090.1 and gi numberGI:190356540  48 AGCCTGCGTGGCGGCCA  49 AGCCUGCGTGGTGCCCUGCC  50CACTAGTUTGGTCGATGAGGCT  51 CCATCTGTAGATTAGGGUAATGAGGCT  52CCCAACTTTGCAAGTGTTGTTCTCGG  53 CCCCAATTTTGCTAGTGTTGTTCTTGG  54CCUGCGTGGCTGCCTGC  55 CGTCGACCGAAGTCCTGCAAAAGGTCAC  56CTGCTGCTGAAAATAGTTCTGTGTTTGG  57 GAAACACGGACACCCAAAGTAGT  58GGCCTCTGCTAAAGCAACACC  59 GTAGATCGGGGCAATGGGGCT  60GTAGATCGGGGTAATGGGGCT  61 GTGGCGCGGGCGAACTGC  62 GTTGCACGGGCGAACTGC  63TACATGCACATCGCCGGGCAGGA  64 TACATGCACATCTCGGGCCAGGA  65TAGACCTGGCAGATGAGGC  66 TGCCGCAGAACGTTGCGAA  67TGCCTCAGGCTTAGGCATAATAGGTATGTATCG  68 TGCTGCAGAAAATAGCTCTGTGTTTGG  69TGCTTCAGGTCTAGGTATAATCGGAATGTACAG  70 TGCTTCAGGTTTAGGCATAATCGGAATGTACAG 71 TGGCCACTCCGTCGATGATG  72 TGGCTACCCCATCGATGATG  73TGGCTACCCCATCGATGCTG  74 TGGCTACCCCUTCGATGATG  75 CTAGTUTGGTCGATGAGGCT 76 Human rhinovirus C isolate Resp_4051/075′ UTR. HM581865.1 GI:302378331  77 CCATCTGTAGRTYRGGGYAATGRGGCTAC  78GTAGATTAGGGUAATGAGGCTAC  79 GTAGATTAGGGUAATGAGGCT  80GTAGATCRGGGUAATGGGGCT  81 GTAGATCRGGGYAATGGGGCT  82GTAGATCRGGGUAATGRGGCT  83 GTAGATCGGGGTAATGGGGCTAC  84GTAGATCGGGGCAATGGGGCTAC  85 GTAGATCRGGGYAATGRGG  86GTAGATCRGGGYAATGRGGCTAC  87 AGCCTGCGTGGYKSCCWRCC  88AGCCYGCGTGGTGCCCYGCC  89 CUGCGTGGTGCCCYGCC  90 CYGCGTGGTGCCCUGCC  91AGCCYGCGTGGTGC  92 AGCCTGCGTGGCGGCCA  93 AGCCYGCGTGGTGCCC  94CCUGCGTGGCTGCCTGC  95 GRTTAGCCRCATTCAGGGGCCGGAGGA  96GAAACACGGACACCCAAAGTAGTYGGTYCCRTCCC  97 AAGTAGTTGGTCCCATCCC  98AAGTAGTTGGTTCCATCCC  99 AAGTAGTCGGTCCCATCCC 100 AAGTAGTTGGTCCCGTCCC 101Human rhinovirus 37 5′ UTR EU096024.1 102 CACTAGTTTGGYCGATGAGGCT 103CTAGTYTGGTCGATGAGGC 104 CTAGTYTGGTCGATGAGG 105 GTYTGGTCGATGAGGC 106TAGTYTGGTCGATGAGGCT 107 TAGTUTGGTCGATGAGGCT 108 RCATTCAGGGGCCGGAGG 109AGCCTGCGTGGCGGCCARCC 110 CCUGCGTGGCTGCCTRC 111 CTGCGTGGTGCCCTACC 112CCYGCGTGGCTGCCTAC 113 AGCCYGCGTGGCTG 114 AGCCYGCGTGGCTGCC 115GRTTAGCCRCATTCRGGRGCCGGAGGA 116 GCATTCAGGGGCCGGAGG 117GAAACACGGACACCCAAAGTAGTYGG 118 GAAACACGGACACCCAAAGTAGTYGGTCC 119AAGTAGTCGGTCCCGTCCC 120 Human rhinovirus A2 EU095989.1 GI:158830711 121YRGRCYTGGCAGATGRGGC 122 TARACCTGGCAGATGRGGC 123 TAGACCTGGCAGATGGGGC 124TAGACCTGGCAGATGRGGC 125 TARACCTGGCAGATGGGGC 126 TARACCTGGTAGATGRGGC 127TARACCTGGCAGATGRG 128 TARACCTGGCAGATGRGG 129 CARACCTGGCAGATGRGGC 130CCTGCCAGATGRGGC 131 WGCCTGCGYGGCKGCCWRC 132 CCYGCGCGGCTGCCTRC 133CCYGCGTGGCTGCCTRC 134 CCYGCGTGGCTGCCTGC 135 WGCCYGCGTGGCTGCCTGC 136AGCCYGCGTGGCTGCCTGC 137 GRTTAGCCGCATTCRGGRGCCGGAGGA 138TACATGCACATCKCSGGVCAGGAYGCYTCGGAG TACCTGAGCCCCG 139ACGCCTCGGAGTACCTGAGCCC 140 CGGGGCTCAGGTACTCCGAGGCGT 141GTKGCRCGGGCRAAYTGCACCA 142 GTKGCRCGGGCGAACTGC 143 GTGGCGCGGGCAAACTG 144GTKGCRCGGGCRAACTGC 145 GTGGCRCGGGCRAACTGC 146 GTKGCGCGGGCRAACTGC 147GTGGCGCGGGCRAACTGC 148 GTGGCRCGGGCGAACTGC 149 GTKGCGCGGGCGAACTGC 150Human metapneumovirus isolate NL/1/99,complete genome. AY525843.1 GI:50059145 151 CSSCCAATTTTGCTAGTGTTGTTCTTGG152 CGGCCAATTTTGCTAGTGTTGTTCTTGG 153 CAATTTTGCTAGTGTTGTTCTTGG 154CCAATTTTGCTAGTGTTGTTCTTGG 155 TGCTTCAGGTYTAGGYATAATCGGAATGTWCAGAGG 156TGCTTCAGGTTTAGGCATAATCGGAATGTTCAGAGG 157 TGCTTCAGGTCTAGGTATAATCGGAATGT158 GCAGAAAATAGCTCTGTGTTTGG 159 Human metapneumovirus isolate SIN05NTU84nucleoprotein (N) gene, partial cds JQ309642.1 GI:374721604 160CTTTGCAAGTGTTGTTCTCGG 161 TGCCTCAGGCYTAGGCATAATMGGYATGTATCGHGGG 162TCAGGCTTAGGCATAATAGGTATGTATCG 163 CCTCAGGCTTAGGCATAATAGGTATGTATCGAG 164CCTCAGGCTTAGGCATAATAGGTATGTATCG 165 CCTCAGGCTTAGGCATAATAGGTATGTATCGCG166 CCTCAGGCTTAGGCATAATAGGTATGTATCGUG 167TGCCTCAGGCCTAGGCATAATCGGCATGTATCGUGGG 168TGCCTCAGGCCTAGGCATAATCGGCATGTATCGTGGG 169CTCAGGCTTAGGCATAATAGGTATGTATCGCG 170 TGCCTCAGGCTTAGGCATAATAGGTATGTATCGC171 TGCCTCAGGCTTAGGCATAATAGGTATGTATCGCG 172TGCCTCAGGCTTAGGCATAATAGGTATGTATCGCGG 173TGCCTCAGGCTTAGGCATAATAGGTATGTATCGUGGG 174TGCCTCAGGCTTAGGCATAATAGGTATGTATCGAGGG 175TGCCTCAGGCTTAGGCATAATCGGTATGTATCGCGGG 176TGCCTCAGGCTTAGGCATAATAGGTATGTATCGCGGG 177 CTGCTGCWGAAAATAGYTCTGTGTTTGG178 TGCTGAAAATAGTTCTGTGTTTGGSequence symbols are per Table 1 of World Intellectual PropertyOrganization (WIPO) Handbook on Industrial Property Information andDocumentation, Standard ST.25 (1998) (“WIPO ST.25 (1998)”).

The contents of the articles, patents, and patent applications, and allother documents and electronically available information mentioned orcited herein, are hereby incorporated by reference in their entirety tothe same extent as if each individual publication was specifically andindividually indicated to be incorporated by reference. Applicantsreserve the right to physically incorporate into this application anyand all materials and information from any such articles, patents,patent applications, or other physical and electronic documents.

The methods illustratively described herein may suitably be practiced inthe absence of any element or elements, limitation or limitations, notspecifically disclosed herein. It is recognized that variousmodifications are possible within the scope of the disclosure claimed.Thus, it should be understood that although the present disclosure hasbeen specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the disclosure embodied thereinherein disclosed may be resorted to by those skilled in the art, andthat such modifications and variations are considered to be within thescope of this disclosure.

The disclosure has been described broadly and generically herein. Eachof the narrower species and subgeneric groupings falling within thegeneric disclosure also form part of the methods. This includes thegeneric description of the methods with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, wherefeatures or aspects of the methods are described in terms of Markushgroups, those skilled in the art will recognize that the disclosure isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

What is claimed is:
 1. A composition or kit comprising at least firstand second amplification oligomers, wherein: the first amplificationoligomer and second amplification oligomer are configured to amplify anAdenovirus amplicon of at least about 50 nucleotides in lengthcomprising at least one Adenovirus position located in the range ofnucleotide positions selected from 52 to 74 and/or 76 to 99 and/or 40 to56 and/or 65 to 87 and/or 1 to 18 and/or 7 to 23 and/or 28 to 45 and/or27 to 45 and/or 26 to 45 and/or 139 to 155 and/or 103 to 123 and/or 159to 175 and/or 83 to 99 and/or 83 to 98 of SEQ ID No.
 47. 2. Acomposition or kit comprising at least first and second amplificationoligomers, wherein: the first amplification oligomer and secondamplification oligomer are configured to amplify a Metapneumovirusamplicon of at least about 50 nucleotides in length comprising at leastone Metapneumovirus position located in the range of nucleotidepositions selected from 966 to 1147 of SEQ ID NO:150, and/or nucleotides844 to 1027 of SEQ ID NO:159, and/or 1000 to 1040 of SEQ ID NO:150,and/or 880 to 915 of SEQ ID NO:159, and/or 1027 to 1080 of SEQ IDNO:150, and/or 913 to 958 of SEQ ID NO:159, and/or 1073 to 1115 of SEQID NO:150, and/or 953 to 995 of SEQ ID NO:159.
 3. A composition or kitcomprising at least first and second amplification oligomers, wherein:the first amplification oligomer and second amplification oligomer areconfigured to amplify a Rhinovirus amplicon of at least about 50nucleotides in length comprising at least one Rhinovirus positionlocated in the range of nucleotide positions selected from 230 to 556 ofSEQ ID NO:120, and/or 199 to 525 of SEQ ID NO:101, and/or 80 to 410 ofSEQ ID NO:76, and/or 263 to 303 of SEQ ID NO:120, and/or 231 to 264 ofSEQ ID NO:101, and/or 106 to 156 of SEQ ID NO:76, and/or 312 to 346 ofSEQ ID NO:120, and/or 279 to 314 of SEQ ID NO:101, and/or 455 to 506 ofSEQ ID NO:76, and/or 480 to 533 of SEQ ID NO:120, and/or 455 to 506 ofSEQ ID NO:101, and/or 338 to 397 of SEQ ID NO:76.
 4. A composition orkit comprising at least first and second amplification oligomersconfigured for two or more target acids, wherein: (A) for a first targetnucleic acid the first amplification oligomer and second amplificationoligomer are configured to amplify an Adenovirus amplicon of at leastabout 50 nucleotides in length comprising at least one Adenovirusposition located in the range of nucleotide positions selected from 52to 74 and/or 76 to 99 and/or 40 to 56 and/or 65 to 87 and/or 1 to 18and/or 7 to 23 and/or 28 to 45 and/or 27 to 45 and/or 26 to 45 and/or139 to 155 and/or 103 to 123 and/or 159 to 175 and/or 83 to 99 and/or 83to 98 of SEQ ID No. 47; and (B) for a second target nucleic acid; (i)the first amplification oligomer and second amplification oligomer areconfigured to amplify a Metapneumovirus amplicon of at least about 50nucleotides in length comprising at least one Metapneumovirus positionlocated in the range of nucleotide positions selected from 966 to 1147of SEQ ID NO:150, and/or nucleotides 844 to 1027 of SEQ ID NO:159,and/or 1000 to 1040 of SEQ ID NO:150, and/or 880 to 915 of SEQ IDNO:159, and/or 1027 to 1080 of SEQ ID NO:150, and/or 913 to 958 of SEQID NO:159, and/or 1073 to 1115 of SEQ ID NO:150, and/or 953 to 995 ofSEQ ID NO:159; or (ii) the first amplification oligomer and secondamplification oligomer are configured to amplify a Rhinovirus ampliconof at least about 50 nucleotides in length comprising at least oneRhinovirus position located in the range of nucleotide positionsselected from 230 to 556 of SEQ ID NO:120, and/or 199 to 525 of SEQ IDNO:101, and/or 80 to 410 of SEQ ID NO:76, and/or 263 to 303 of SEQ IDNO:120, and/or 231 to 264 of SEQ ID NO:101, and/or 106 to 156 of SEQ IDNO:76, and/or 312 to 346 of SEQ ID NO:120, and/or 279 to 314 of SEQ IDNO:101, and/or 455 to 506 of SEQ ID NO:76, and/or 480 to 533 of SEQ IDNO:120, and/or 455 to 506 of SEQ ID NO:101, and/or 338 to 397 of SEQ IDNO:76.
 5. A composition or kit comprising at least first and secondamplification oligomers configured for two or more target acids,wherein: (A) for a first target nucleic acid the first amplificationoligomer and second amplification oligomer are configured to amplify aMetapneumovirus amplicon of at least about 50 nucleotides in lengthcomprising at least one Metapneumovirus position located in the range ofnucleotide positions selected from 966 to 1147 of SEQ ID NO:150, and/ornucleotides 844 to 1027 of SEQ ID NO:159, and/or 1000 to 1040 of SEQ IDNO:150, and/or 880 to 915 of SEQ ID NO:159, and/or 1027 to 1080 of SEQID NO:150, and/or 913 to 958 of SEQ ID NO:159, and/or 1073 to 1115 ofSEQ ID NO:150, and/or 953 to 995 of SEQ ID NO:159; and (B) for a secondtarget nucleic acid; (i) the first amplification oligomer and secondamplification oligomer are configured to amplify an Adenovirus ampliconof at least about 50 nucleotides in length comprising at least oneAdenovirus position located in the range of nucleotide positionsselected from 52 to 74 and/or 76 to 99 and/or 40 to 56 and/or 65 to 87and/or 1 to 18 and/or 7 to 23 and/or 28 to 45 and/or 27 to 45 and/or 26to 45 and/or 139 to 155 and/or 103 to 123 and/or 159 to 175 and/or 83 to99 and/or 83 to 98 of SEQ ID No. 47; or (ii) the first amplificationoligomer and second amplification oligomer are configured to amplify aRhinovirus amplicon of at least about 50 nucleotides in lengthcomprising at least one Rhinovirus position located in the range ofnucleotide positions selected from 230 to 556 of SEQ ID NO:120, and/or199 to 525 of SEQ ID NO:101, and/or 80 to 410 of SEQ ID NO:76, and/or263 to 303 of SEQ ID NO:120, and/or 231 to 264 of SEQ ID NO:101, and/or106 to 156 of SEQ ID NO:76, and/or 312 to 346 of SEQ ID NO:120, and/or279 to 314 of SEQ ID NO:101, and/or 455 to 506 of SEQ ID NO:76, and/or480 to 533 of SEQ ID NO:120, and/or 455 to 506 of SEQ ID NO:101, and/or338 to 397 of SEQ ID NO:76.
 6. A composition or kit comprising at leastfirst and second amplification oligomers configured for two or moretarget acids, wherein: (A) for a first target nucleic acid the firstamplification oligomer and second amplification oligomer are configuredto amplify a Rhinovirus amplicon of at least about 50 nucleotides inlength comprising at least one Rhinovirus position located in the rangeof nucleotide positions selected from 230 to 556 of SEQ ID NO:120,and/or 199 to 525 of SEQ ID NO:101, and/or 80 to 410 of SEQ ID NO:76,and/or 263 to 303 of SEQ ID NO:120, and/or 231 to 264 of SEQ ID NO:101,and/or 106 to 156 of SEQ ID NO:76, and/or 312 to 346 of SEQ ID NO:120,and/or 279 to 314 of SEQ ID NO:101, and/or 455 to 506 of SEQ ID NO:76,and/or 480 to 533 of SEQ ID NO:120, and/or 455 to 506 of SEQ ID NO:101,and/or 338 to 397 of SEQ ID NO:76; and (B) for a second target nucleicacid; (i) the first amplification oligomer and second amplificationoligomer are configured to amplify an Adenovirus amplicon of at leastabout 50 nucleotides in length comprising at least one Adenovirusposition located in the range of nucleotide positions selected from 52to 74 and/or 76 to 99 and/or 40 to 56 and/or 65 to 87 and/or 1 to 18and/or 7 to 23 and/or 28 to 45 and/or 27 to 45 and/or 26 to 45 and/or139 to 155 and/or 103 to 123 and/or 159 to 175 and/or 83 to 99 and/or 83to 98 of SEQ ID No. 47; or (ii) the first amplification oligomer andsecond amplification oligomer are configured to amplify aMetapneumovirus amplicon of at least about 50 nucleotides in lengthcomprising at least one Metapneumovirus position located in the range ofnucleotide positions selected from 966 to 1147 of SEQ ID NO:150, and/ornucleotides 844 to 1027 of SEQ ID NO:159, and/or 1000 to 1040 of SEQ IDNO:150, and/or 880 to 915 of SEQ ID NO:159, and/or 1027 to 1080 of SEQID NO:150, and/or 913 to 958 of SEQ ID NO:159, and/or 1073 to 1115 ofSEQ ID NO:150, and/or 953 to 995 of SEQ ID NO:159.
 7. A composition orkit comprising at least first and second amplification oligomersconfigured for three or more target acids, wherein: (A) for a firsttarget nucleic acid the first amplification oligomer and secondamplification oligomer are configured to amplify an Adenovirus ampliconof at least about 50 nucleotides in length comprising at least oneAdenovirus position located in the range of nucleotide positionsselected from 52 to 74 and/or 76 to 99 and/or 40 to 56 and/or 65 to 87and/or 1 to 18 and/or 7 to 23 and/or 28 to 45 and/or 27 to 45 and/or 26to 45 and/or 139 to 155 and/or 103 to 123 and/or 159 to 175 and/or 83 to99 and/or 83 to 98 of SEQ ID No. 47; and (B) for a second target nucleicacid the first amplification oligomer and second amplification oligomerare configured to amplify a Metapneumovirus amplicon of at least about50 nucleotides in length comprising at least one Metapneumovirusposition located in the range of nucleotide positions selected from 966to 1147 of SEQ ID NO:150, and/or nucleotides 844 to 1027 of SEQ IDNO:159, and/or 1000 to 1040 of SEQ ID NO:150, and/or 880 to 915 of SEQID NO:159, and/or 1027 to 1080 of SEQ ID NO:150, and/or 913 to 958 ofSEQ ID NO:159, and/or 1073 to 1115 of SEQ ID NO:150, and/or 953 to 995of SEQ ID NO:159; and (C) for a third target nucleic acid the firstamplification oligomer and second amplification oligomer are configuredto amplify a Rhinovirus amplicon of at least about 50 nucleotides inlength comprising at least one Rhinovirus position located in the rangeof nucleotide positions selected from 230 to 556 of SEQ ID NO:120,and/or 199 to 525 of SEQ ID NO:101, and/or 80 to 410 of SEQ ID NO:76,and/or 263 to 303 of SEQ ID NO:120, and/or 231 to 264 of SEQ ID NO:101,and/or 106 to 156 of SEQ ID NO:76, and/or 312 to 346 of SEQ ID NO:120,and/or 279 to 314 of SEQ ID NO:101, and/or 455 to 506 of SEQ ID NO:76,and/or 480 to 533 of SEQ ID NO:120, and/or 455 to 506 of SEQ ID NO:101,and/or 338 to 397 of SEQ ID NO:76.
 8. The composition or kit of any oneof claims 1 to 3, wherein the first amplification oligomer comprises anucleic acid sequences that contains at least one 5-Me-dC, at least onenon-Watson Crick base, at least one degenerate base, or a combinationthereof.
 9. The composition or kit of any of claim 1 to 3 or 8, whereinthe second amplification oligomer comprises a nucleic acid sequence thatcontains at least one 5-Me-dC, or at least one non-Watson Crick base, orat least one degenerate base, or a combination thereof.
 10. Thecomposition or kit of claim 4 or claim 7, wherein the firstamplification oligomer configured to amplify an Adenovirus ampliconcomprises a nucleic acid sequences that contains at least one 5-Me-dC,or at least one non-Watson Crick base, or at least one degenerate base,or a combination thereof, and/or wherein the second amplificationoligomer configured to amplify an Adenovirus amplicon comprises anucleic acid sequences that contains at least one 5-Me-dC, or at leastone non-Watson Crick base, or at least one degenerate base, or acombination thereof.
 11. The composition or kit of claim 5 or claim 6,wherein the second target nucleic acid is an Adenovirus target nucleicacid and wherein the first amplification oligomer configured to amplifyan Adenovirus amplicon comprises a nucleic acid sequences that containsat least one 5-Me-dC, at least one non-Watson Crick base, at least onedegenerate base, or a combination thereof, or wherein the secondamplification oligomer configured to amplify an Adenovirus ampliconcomprises a nucleic acid sequences that contains at least one 5-Me-dC,at least one non-Watson Crick base, at least one degenerate base, or acombination thereof, or both.
 12. The composition or kit of claim 5 orclaim 7, wherein the first amplification oligomer configured to amplifya Metapneumovirus amplicon comprises a nucleic acid sequences thatcontains at least one 5-Me-dC, at least one non-Watson Crick base, atleast one degenerate base, or a combination thereof, or wherein thesecond amplification oligomer configured to amplify an Metapneumovirusamplicon comprises a nucleic acid sequences that contains at least one5-Me-dC, at least one non-Watson Crick base, at least one degeneratebase, or a combination thereof, or both.
 13. The composition or kit ofclaim 4 or claim 6, wherein the second target nucleic acid is anMetapneumovirus target nucleic acid and wherein the first amplificationoligomer configured to amplify an Metapneumovirus amplicon comprises anucleic acid sequences that contains at least one 5-Me-dC, at least onenon-Watson Crick base, at least one degenerate base, or a combinationthereof, or wherein the second amplification oligomer configured toamplify an Metapneumovirus amplicon comprises a nucleic acid sequencesthat contains at least one 5-Me-dC, at least one non-Watson Crick base,at least one degenerate base, or a combination thereof, or both.
 14. Thecomposition or kit of claim 6 or claim 7, wherein the firstamplification oligomer configured to amplify a Rhinovirus ampliconcomprises a nucleic acid sequences that contains at least one 5-Me-dC,at least one non-Watson Crick base, at least one degenerate base, or acombination thereof, or wherein the second amplification oligomerconfigured to amplify an Rhinovirus amplicon comprises a nucleic acidsequences that contains at least one 5-Me-dC, at least one non-WatsonCrick base, at least one degenerate base, or a combination thereof, orboth.
 15. The composition or kit of claim 4 or claim 5, wherein thesecond target nucleic acid is an Rhinovirus target nucleic acid andwherein the first amplification oligomer configured to amplify anRhinovirus amplicon comprises a nucleic acid sequences that contains atleast one 5-Me-dC, at least one non-Watson Crick base, at least onedegenerate base, or a combination thereof, or wherein the secondamplification oligomer configured to amplify an Rhinovirus ampliconcomprises a nucleic acid sequences that contains at least one 5-Me-dC,at least one non-Watson Crick base, at least one degenerate base, or acombination thereof, or both.
 16. The composition or kit of any one ofclaim 1, 4, 7, 8, 10 or 11, wherein for the Adenovirus target nucleicacid the first amplification oligomer comprises a target hybridizingsequence selected from the group consisting of SEQ ID NOS: 1, 5, 11, 12,25, 26, 31, 32, 33, 34, 35, 38, 71, 72, 73,
 74. 17. The composition orkit of claim 5, 6 or 9, wherein the second target nucleic acid isAdenovirus and wherein the first amplification oligomer comprises atarget hybridizing sequence selected from the group consisting of SEQ IDNOS: 1, 5, 11, 12, 25, 26, 31, 32, 33, 34, 35, 38, 71, 72, 73,
 74. 18.The composition or kit of any one of claim 1, 4, 7, 8, 10 or 11, whereinfor the Adenovirus target nucleic acid the second amplification oligomercomprises a target hybridizing sequence selected from the groupconsisting of SEQ ID NOS: 2, 3, 6, 7, 8, 9, 13, 14, 15, 16, 27, 28, 42,43, 44, 45, 46, 61, 62, 138, 139, 140, 141, 142, 143, 144, 145, 146,147, 148,
 149. 19. The composition or kit of claim 5, 6 or 9, whereinthe second target nucleic acid is Adenovirus and wherein the secondamplification oligomer comprises a target hybridizing sequence selectedfrom the group consisting of SEQ ID NOS: 2, 3, 6, 7, 8, 9, 13, 14, 15,16, 27, 28, 42, 43, 44, 45, 46, 61, 62, 138, 139, 140, 141, 142, 143,144, 145, 146, 147, 148,
 149. 20. The composition or kit of any one ofclaim 2, 5, 7, 8, 12 or 13, wherein for the Metapneumovirus targetnucleic acid the first amplification oligomer comprises, consists ofconsists essentially of a target hybridizing sequence selected from thegroup consisting of SEQ ID NOS:52, 53, 151, 152, 153, 154,
 160. 21. Thecomposition or kit of claim 4, 6, or 9, wherein the second targetnucleic acid is Metapneumovirus and wherein the first amplificationoligomer comprises, consists of consists essentially of a targethybridizing sequence selected from the group consisting of SEQ IDNOS:52, 53, 151, 152, 153, 154,
 160. 22. The composition or kit of anyone of claim 2, 5, 7, 8, 12 or 13, wherein for the Metapneumovirustarget nucleic acid the second amplification oligomer comprises,consists of consists essentially of a target hybridizing sequenceselected from the group consisting of SEQ ID NOS:56, 68, 158, 177, 178.23. The composition or kit of claim 4, 6, or 9, wherein the secondtarget nucleic acid is Metapneumovirus and wherein the secondamplification oligomer comprises, consists of consists essentially of atarget hybridizing sequence selected from the group consisting of SEQ IDNOS:56, 68, 158, 177,
 178. 24. The composition or kit of any one ofclaim 3, 6, 7, 8, 14 or 15, wherein for the Rhinovirus target nucleicacid the first amplification oligomer comprises, consists of consistsessentially of a target hybridizing sequence selected from the groupconsisting of SEQ ID NOS:50, 51, 59, 60, 65, 75, 77 to 86, 102 to 108,121 to
 130. 25. The composition or kit of claim 4, 5, or 9, wherein thesecond target nucleic acid is Rhinovirus and wherein the firstamplification oligomer comprises, consists of consists essentially of atarget hybridizing sequence selected from the group consisting of SEQ IDNOS:50, 51, 59, 60, 65, 75, 77 to 86, 102 to 108, 121 to
 130. 26. Thecomposition or kit of any one of claim 3, 6, 7, 8, 14 or 15, wherein forthe Rhinovirus target nucleic acid the second amplification oligomercomprises, consists of consists essentially of a target hybridizingsequence selected from the group consisting of SEQ ID NOS:57, 95 to 100,115 to 119,
 137. 27. The composition or kit of claim 4, 5, or 9, whereinthe second target nucleic acid is Rhinovirus and wherein the secondamplification oligomer comprises, consists of consists essentially of atarget hybridizing sequence selected from the group consisting of SEQ IDNOS:57, 95 to 100, 115 to 119,
 137. 28. The composition or kit of anyone of claims 1 to 27, wherein the composition or kit further comprisesa least one detection probe oligomer.
 29. The composition or kit of anyone of claims 1, 4, 7, 8 to 11, and 16 to 19, wherein the composition orkit further comprises an Adenovirus detection probe oligomer comprisinga sequence selected from the group consisting of SEQ ID NOS:4, 10, 17,18, 19, 20, 21, 22, 23, 24, 29, 30, 36, 37, 39, 40, 63, 64, 139, 140.30. The composition or kit of any one of claim 5 or 6, wherein thesecond target nucleic acid is Adenovirus and wherein the composition orkit further comprises a detection probe oligomer comprising a sequenceselected from the group consisting of SEQ ID Nos. 4, 10, 17, 18, 19, 20,21, 22, 23, 24, 29, 30, 36, 37, 39, 40, 63, 64, 139,
 140. 31. Thecomposition or kit of any one of claims 1, 4, 7, 8 to 11, and 16 to 19,wherein the composition or kit further comprises an Adenovirus detectionprobe oligomer comprising a sequence that is from 18 to 36 nucleobasesin length wherein the 18 to 36 nucleobases are all selected fromcontiguous nucleobases within SEQ ID NO:138.
 32. The composition or kitof any one of claim 15 or 6, wherein the second target nucleic acid isAdenovirus and wherein the composition or kit further comprises adetection probe oligomer comprising a sequence that is from 18 to 36nucleobases in length wherein the 18 to 36 nucleobases are all selectedfrom contiguous nucleobases within SEQ ID NO:138.
 33. The composition orkit of any one of claims 2, 5, 7, 8, 9, 12, 13, 20, 21, 22, and 23,wherein the composition or kit further comprises a Metapneumovirusdetection probe oligomer comprising a sequence selected from the groupconsisting of SEQ ID Nos. 67, 69, 70, 155, 156, 157, 161, 162, 163, 164,165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, and
 176. 34. Thecomposition or kit of any one of claim 4 or 6, wherein the second targetnucleic acid is Metapneumovirus and wherein the composition or kitfurther comprises a detection probe oligomer comprising a sequenceselected from the group consisting of SEQ ID Nos. 67, 69, 70, 155, 156,157, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173,174, 175, and
 176. 35. The composition or kit of any one of claims 2, 5,7, 8, 9, 12, 13, 20, 21, 22, and 23, wherein the composition or kitfurther comprises a Metapneumovirus detection probe oligomer comprisinga sequence that is from 18 to 36 nucleobases in length wherein the 18 to36 nucleobases are all selected from contiguous nucleobases within SEQID NO:161 or within SEQ ID NO:155.
 36. The composition or kit of any oneof claim 4 or 6, wherein the second target nucleic acid isMetapneumovirus and wherein the composition or kit further comprises adetection probe oligomer comprising a sequence that is from 18 to 36nucleobases in length wherein the 18 to 36 nucleobases are all selectedfrom contiguous nucleobases within SEQ ID NO:161 or within SEQ IDNO:155.
 37. The composition or kit of any one of claims 3, 6, 7, 8, 9,14, 15, and 24 to 27, wherein the composition or kit further comprises aRhinovirus detection probe oligomer comprising a sequence selected fromthe group consisting of SEQ ID Nos. 48, 49, 54, 87 to 94, 109 to 114,and 131 to
 136. 38. The composition or kit of any one of claim 4 or 5,wherein the second target nucleic acid is Rhinovirus and wherein thecomposition or kit further comprises a detection probe oligomercomprising a sequence selected from the group consisting of SEQ ID Nos.48, 49, 54, 87 to 94, 109 to 114, 131 to
 136. 39. The composition or kitof any one of claims 28 to 38, wherein at least one of the detectionprobe oligomers comprises at least one 5-Me-dC, or at least onenon-Watson Crick base, or at least one of a degenerate base, or acombination thereof.
 40. The composition or kit of any one of claims 28to 39, wherein at least one of the detection probe oligomers comprises adetectable label.
 41. The composition or kit of claim 40, wherein thedetectable label is a fluorophore.
 42. The composition or kit of claim40 or claim 41, wherein the detection probe oligomer is a dual labeleddetection probe oligomer.
 43. The composition or kit of claim 42,wherein the detection probe oligomer comprises a fluorescent detectablelabel and a quencher moiety that can quench a fluorescent emission fromthe fluorescent label.
 44. The composition or kit of any one of claims1, 4, 7 to 11, 16 to 19, 28 to 32, and 39 to 43, wherein the compositionor kit further comprises one or more additional amplification oligomerseach of which is configured to amplify an Adenovirus target nucleicacid.
 45. The composition or kit of claim 5 or claim 6, wherein thesecond target nucleic acid is Adenovirus and wherein the composition orkit further comprises one or more additional amplification oligomerseach of which is configured to amplify an Adenovirus target nucleicacid.
 46. The composition or kit of claim 44 or 45, wherein each of theone or more additional amplification oligomers comprise a targethybridizing sequence separately selected from the group consisting ofSEQ ID NOS: 1 to 9, 11 to 16, 25 to 28, 31 to 35, 38, 42 to 46, 61, 62,and 71 to
 74. 47. The composition or kit of any one of claims 2, 5, 7,8, 9, 12, 13, 20 to 23, 28, 33 to 36, and 39 to 43, wherein thecomposition or kit further comprises one or more additionalamplification oligomers each of which is configured to amplify aMetapneumovirus target nucleic acid.
 48. The composition or kit of claim4 or claim 6, wherein the second target nucleic acid is Metapneumovirusand wherein the composition or kit further comprises one or moreadditional amplification oligomers each of which is configured toamplify a Metapneumovirus target nucleic acid.
 49. The composition orkit of claim 47 or 48, wherein each of the one or more additionalamplification oligomers comprise a target hybridizing sequenceseparately selected from the group consisting of SEQ ID NOS:52, 53, 56,68, 151, 152, 153, 154, 158, 160, 177,
 178. 50. The composition or kitof any one of claims 3, 6, 7, 8, 9, 14, 15, 24 to 28, and 37 to 43,wherein the composition or kit further comprises one or more additionalamplification oligomers each of which is configured to amplify aRhinovirus target nucleic acid.
 51. The composition or kit of claim 4 orclaim 5, wherein the second target nucleic acid is Rhinovirus andwherein the composition or kit further comprises one or more additionalamplification oligomers each of which is configured to amplify aRhinovirus target nucleic acid.
 52. The composition or kit of claim 47or 48, wherein each of the one or more additional amplificationoligomers comprise a target hybridizing sequence separately selectedfrom the group consisting of SEQ ID NOS:50, 51, 57, 59, 60, 65, 75, 77to 86, 95 to 100, 102 to 108, 115 to 119, 121 to 130,
 137. 53. Thecomposition or kit of any one of claims 1, 4, 7 to 11, 16 to 19, 28 to32, and 39 to 47, wherein the composition or kit comprises at leastfirst and second amplification oligomers and one or more additionalamplification oligomers configured to amplify an Adenovirus targetnucleic acid each of the amplification oligomers independentlycomprising a sequences selected from the group consisting of: SEQ IDNOS:61, 62, 71, 72, 73, and
 74. 54. The composition or kit of any one ofclaims 2, 5, 7, 8, 9, 12, 13, 20 to 23, 28, 33 to 36, 39 to 43, 46 to49, and 53, wherein the composition or kit comprises at least first andsecond amplification oligomers and one or more additional amplificationoligomers configured to amplify a Metapneumovirus target nucleic acideach of the amplification oligomers each independently comprising asequences selected from the group consisting of: SEQ ID NOS:52, 53, 56,and
 58. 55. The composition or kit of any one of claims 3, 6, 7, 8, 9,14, 15, 24 to 28, 37 to 43, and 50 to 54, wherein the composition or kitcomprises at least first and second amplification oligomers and one ormore additional amplification oligomers configured to amplify aRhinovirus target nucleic acid each of the amplification oligomers eachindependently comprising a sequences selected from the group consistingof: SEQ ID NOS:50, 51, 57, 59, 60, and
 65. 56. The composition or kit ofany one of claims 1, 4, 7 to 11, 16 to 19, 28 to 32, 39 to 47 and 53 to55, wherein the composition or kit further comprises two Adenovirusdetection probe oligomers, each of the detection probe oligomersindependently comprising a sequence selected from the group consistingof SEQ ID NOS:63 and
 64. 57. The composition or kit of any one of claims2, 5, 7, 8, 9, 12, 13, 20 to 23, 28, 33 to 36, 39 to 43, 46 to 49, and53 to 56, wherein the composition or kit further comprises threeMetapneumovirus detection probe oligomers, each of the detection probeoligomers independently comprising a sequence selected from the groupconsisting of SEQ ID NOS:67, 69 and
 70. 58. The composition or kit ofany one of claims 3, 6, 7, 8, 9, 14, 15, 24 to 28, 37 to 43 and 50 to57, wherein the composition or kit further comprises three Rhinovirusdetection probe oligomers, each of the detection probe oligomersindependently comprising a sequence selected from the group consistingof SEQ ID NOS:48, 49 and
 54. 59. The composition or kit of any one ofthe preceding claims, wherein the composition or kit further comprises anucleic acid target capture probe comprising a target hybridizingsequence and an immobilized probe binding region.
 60. The composition orkit of claim 59, wherein the target hybridizing sequence is a poly-Knucleotide sequence.
 61. The composition or kit of claim 60, wherein thepoly-K nucleotide sequence is a random poly-GU sequence.
 62. Thecomposition or kit of claim 59, 60 or 61, wherein immobilized probebinding region is a homopolymeric nucleotide sequence, preferablycomprising a nucleotide sequence selected from the group consisting ofT₀₋₄A₁₀₋₃₆.
 63. The composition or kit of any one of claims 1 to 62,wherein the composition further comprises an enzyme, a buffer, dNTPs, ora combination thereof.
 64. A method for the determining the presence ofabsence of an Adenovirus target nucleic acid, a Metapneumovirus targetnucleic acid, a Rhinovirus target nucleic acid, or a combination thereofin a sample, the method comprising the steps of: (A) contacting a samplewith a combination of amplification oligomers from any one of claims 1to 58; (B) performing an in vitro nucleic acid amplification reactionwherein any of an Adenovirus target nucleic acid, a Metapneumovirustarget nucleic acid, or a Rhinovirus target nucleic acid in the sampleis used by the combination of amplification oligomers configured toamplify that target nucleic acid to generate an amplification product;and (C) detecting the amplification product; thereby determining thepresence or absence of the target nucleic acid in the sample.
 65. Themethod of claim 64, wherein the sample is a sample derived from a human.66. The method of claim 65, wherein the sample is a mucosal sample. 67.The method of claim 65 or claim 66, wherein the sample is obtained usinga nasopharyngeal swab.
 68. The method of any one of claims 64 to 67,wherein, before step (A) a sample preparation step is performed toseparate any target nucleic acid in the sample away from other samplecomponents.
 69. The method of claim 68, wherein the sample preparationstep comprises a target capture step.
 70. The method of claim 69,wherein the target capture step comprise contacting the sample with anucleic acid target capture probe comprising a target hybridizingsequence and an immobilized probe binding region.
 71. The method ofclaim 70, wherein the target hybridizing sequence is a poly-K nucleotidesequence.
 72. The method of claim 71, wherein the poly-K nucleotidesequence is a random poly-GU sequence.
 73. The method of claim 70, 71 or72, wherein immobilized probe binding region is a homopolymericnucleotide sequence, preferably comprising a nucleotide sequenceselected from the group consisting of T₀₋₄A₁₀₋₃₆.
 74. The method of anyone of claims 64 to 73, wherein the detecting step (C) is performedusing one or more detection probe oligomers.
 75. The method of claim 74,wherein each of the one or more detection probe oligomers areindividually selected from the group consisting of: SEQ ID NOS:4, 10,17, 18, 19, 20, 21, 22, 23, 24, 29, 30, 36, 37, 39, 40, 63, 64, 139,140, 67, 69, 70, 155, 156, 157, 161, 162, 163, 164, 165, 166, 167, 168,169, 170, 171, 172, 173, 174, 175, 176, 48, 49, 54, 87 to 94, 109 to114, and 131 to
 136. 76. The method of claim 74 or 75, wherein at leastone of the detection probe oligomers comprises at least one 5-Me-dC, orat least one non-Watson Crick base, or at least one of a degeneratebase, or a combination thereof.
 77. The method of claim 74, 75 or 76,wherein at least one of the detection probe oligomers comprises adetectable label.
 78. The method of claim 77, wherein the detectablelabel is a fluorophore.
 79. The method of claim 77 or claim 78, whereinthe detection probe oligomer is a dual labeled detection probe oligomer.80. The method of claim 79, wherein the detection probe oligomercomprises a fluorescent detectable label and a quencher moiety that canquench a fluorescent emission from the fluorescent label.
 81. The methodof any one of claims 64 to 80, wherein the in vitro nucleic acidamplification reaction comprises thermal cycling.
 82. The method of anyone of claims 64 to 81, wherein the in vitro nucleic acid amplificationreaction comprises PCR with a polymerase enzyme having 5′ to 3′exonuclease activity.
 83. The method of any one of claims 75 to 80,wherein the in vitro nucleic acid amplification reaction is performedusing an enzyme having 5′ to 3′ exonuclease activity.
 84. The method ofany one of claims 77 to 80, wherein the in vitro nucleic acidamplification reaction is performed using an enzyme having 5′ to 3′exonuclease activity and wherein an amplification product is detected bydetermining a fluorescence value that is above a predetermined thresholdvalue.
 85. A system for performing one or more steps of the method ofclaim 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, or
 84. 86. The system of claim 85, wherein the system isan automated system.
 87. The system of claim 85 or 86, wherein thesystem performs all of the steps of the method.
 88. A method for the invitro detection of an Adenovirus target nucleic acid, a Metapneumovirustarget nucleic acid, a Rhinovirus target nucleic acid, or a combinationthereof in a sample, wherein the method comprises contacting anAdenovirus target nucleic acid, a Metapneumovirus target nucleic acid,and/or a Rhinovirus target nucleic acid with a detection probe oligomerfrom any of claims 29 to 43, wherein hybridization between the detectionprobe oligomers and the target nucleic acid to which the detection probeoligomer is configured to hybridize indicates the presence of thattarget nucleic acid.
 89. The method of claim 88, wherein the methodcomprises contacting an amplification product from the Adenovirus targetnucleic acid, the Metapneumovirus target nucleic acid, and/or theRhinovirus target nucleic acid with the detection probe oligomer,wherein hybridization between the detection probe oligomers and theamplification product to which the detection probe oligomer isconfigured to hybridize indicates the presence of that target nucleicacid from which the amplification product was generated.
 90. The methodof claim 88 or 89, wherein the in vitro detection reaction is performedusing an enzyme having 5′ to 3′ exonuclease activity.
 91. The method ofany one of claims 88 to 90, wherein the in vitro detection reaction isperformed using an enzyme having 5′ to 3′ exonuclease activity andwherein the target nucleic acid or the amplification product generatedtherefrom is detected by determining a fluorescence value that is abovea predetermined threshold value.
 92. A system for performing the invitro detection reaction of any one of claims 88 to
 91. 93. The systemof claim 92, wherein the system is an automated system.
 94. The systemof claim 92 or 93, wherein the system performs all of the steps of themethod.
 95. A dried composition comprising one or more of theamplification oligomers from any one of claims 1 to
 27. 96. A driedcomposition comprising one or more of the amplification oligomers fromany one of claims 44 to
 55. 97. A dried composition comprising one ormore of the detection probe oligomers from any of claims 29 to 43 or 56to
 58. 98. A dried composition comprising a combination of amplificationoligomers and/or detection probe oligomers from any one of claims 1 to58.
 99. The dried composition of any one of claims 95 to 98, wherein thedried composition further comprises an enzyme, dNTPs, or both.
 100. Thedried composition of claim 99, wherein the enzyme having 5′ to 3′exonuclease activity.
 101. The dried composition of claim 99 or claim100, wherein the enzyme is a polymerase enzyme.
 102. The driedcomposition of any one of claims 95 to 101, wherein the driedcomposition has an inorganic salt concentration of 10 mM or less. 103.The dried composition of any one of claims 95 to 102, wherein the driedcomposition has an inorganic salt concentration of 7 mM or less. 104.The dried composition of any one of claims 95 to 103, wherein the driedcomposition has an inorganic salt concentration of 5 mM or less. 105.The dried composition of any one of claims 95 to 101, wherein the driedcomposition has an inorganic salt concentration of between about 0.5 mMto about 10 mM.